Injectable vaccines against multiple meningococcal serogroups

ABSTRACT

An injectable immunogenic composition comprising capsular saccharides from at least two of serogroups A, C, W135 and Y of Neisseria meningitidis, wherein said capsular saccharides are conjugated to carrier protein(s) and/or are oligosaccharides, and wherein (i) the composition comprises &lt;50 μg meningococcal saccharide per dose, and/or (ii) the composition further comprises an antigen from one or more of: (a) serogroup B N.meningitidis; (b) Haemophilus influenzae type B; and/or (c) Streptococcus pneumoniae. Saccharide antigens in the compositions are generally conjugated to a carrier.

SEROGROUPS CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.15/240,385, filed Aug. 18, 2016, which is the Divisional of U.S. patentapplication Ser. No. 10/543,455, which claims an international filingdate of Jan. 30, 2004, which is the National Stage of InternationalPatent Application No. PCT/IB2004/000651, filed Jan. 30, 2004, whichclaims the benefit of United Kingdom Patent Application No. 0323101.6,filed Oct. 2, 2003, and United Kingdom Patent Application No. 0302217.5,filed Jan. 30, 2003, each of which is hereby incorporated by referencein its entirety.

SUBMISSION OF′ SEQUENCE LISTING AS ASCHII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 303822006710SEQUST.TXT,date recorded: Aug. 17, 2016, size: 31 KB).

TECHNICAL FIELD

This invention is in the field of vaccines, particularly againstbacterial meningitis.

BACKGROUND ART

Neisseria meningitidis is a Gram-negative human pathogen [1] whichcauses bacterial meningitis. It is closely related to N. gonorrhoeae,although one feature that clearly differentiates meningococcus is thepresence of a polysaccharide capsule that is present in all pathogenicmeningococci.

Based on the organism's capsular polysaccharide, twelve serogroups of N.meningitidis have been identified (A, B, C, H, I, K, L, 29E, W135, X, Y& Z). Serogroup A (‘MenA’) is most common cause of epidemic disease insub-Saharan Africa. Serogroups B & C are responsible for the majority ofcases in developed countries, with the remaining cases being caused byserogroups W135 & Y.

As well as being used for classification, the capsular polysaccharidehas been used for vaccination. An injectable tetravalent vaccine ofcapsular polysaccharides from serogroups A, C, Y & W135 has been knownfor many years [2,3] and is licensed for human use. Although effectivein adolescents and adults, it induces a poor immune response and shortduration of protection and cannot be used in infants [e.g. 4]. Thepolysaccharides in this vaccine are unconjugated and are present at a1:1:1:1 weight ratio [5]. Mencevax ACWY™ and Menomune™ both contain 50μg of each purified polysaccharide once reconstituted from theirlyophilised forms. The capsular saccharides of serogroups A, C, W135 & Yhave also been combined in the form of conjugates [6-9] to givetetravalent vaccines e.g. the unadjuvanted Menactra™ product.

Conjugated serogroup C oligosaccharides have been approved for humanuse, including Menjugate™ [10,11], Meningitec™ and NeisVac-C™. TheMenjugate™ and Meningitec™ products have oligosaccharide antigensconjugated to a CRM₁₉₇ carrier, whereas NeisVac-C™ uses the completepolysaccharide (de-O-acetylated) conjugated to a tetanus toxoid carrier.

There remains, however, a need for improvements in conjugate vaccinesagainst serogroups A, W135 and Y, and in their manufacture. That need isaddressed by the products, processes and uses disclosed in reference 7,but there remains scope for further modifications and improvements.

DISCLOSURE OF THE INVENTION

The invention provides an injectable immunogenic composition comprisingcapsular saccharides from at least two of serogroups A, C, W135 and Y ofN.meningitidis, wherein said capsular saccharides are conjugated tocarrier protein(s) and/or are oligosaccharides, and wherein thecomposition comprises ≤50 μg meningococcal saccharide per dose.

The invention also provides an injectable immunogenic compositioncomprising capsular saccharides from at least two of serogroups A, C,W135 and Y of N.meningitidis, wherein said capsular saccharides areconjugated to carrier protein(s) and/or are oligosaccharides, andwherein the composition further comprises an antigen from one or moreof: (a) serogroup B N. meningitidis; (b) Haemophilus influenzae type B;and/or (c) Streptococcus pneumoniae.

Antigens included in sixteen preferred compositions of the inventionare: (1) serogroups C, W135 & Y of N.meningitidis; (2) serogroups A, C,W135 & Y of N.meningitidis; (3) serogroups B, C, W135 & Y ofN.meningitidis; (4) serogroups A, B, C, W135 & Y of N.meningitidis; (5)H.influenzae type B and serogroups C, W135 & Y of N.meningitidis; (6)H.influenzae type B and serogroups A, C, W135 & Y of N.meningitidis; (7)H.influenzae type B and serogroups B, C, W135 & Y of N.meningitidis; (8)H.influenzae type B and serogroups A, B, C, W135 & Y of N.meningitidis;(9) S.pneumoniae and serogroups C, W135 & Y of N.meningitidis; (10)S.pneumoniae and serogroups A, C, W135 & Y of N.meningitidis; (11)S.pneumoniae and serogroups B, C, W135 & Y of N.meningitidis; (12)S.pneumoniae and serogroups A, B, C, W135 & Y of N.meningitidis; (13)H.influenzae type B, S.pneumoniae and serogroups C, W135 & Y ofN.meningitidis; (14) H.influenzae type B, S.pneumoniae and serogroups A,C, W135 & Y of N.meningitidis; (15) H.influenzae type B, S.pneumoniaeand serogroups B, C, W135 & Y of N.meningitidis; (16) H.influenzae typeB, S.pneumoniae and serogroups A, B, C, W135 & Y of N.meningitidis.

Saccharide antigens in the compositions of the invention are preferablyconjugated to a carrier. Saccharide antigens in the compositions of theinvention are preferably oligosaccharides. Saccharide antigens in thecompositions of the invention are most preferably conjugatedoligosaccharides.

Meningococcal Saccharide Mixtures

The compositions of the invention comprise capsular saccharides from atleast two (i.e. 2, 3 or 4) of serogroups A, C, W135 and Y ofN.meningitidis. Compositions of the invention preferably includeN.meningitidis saccharides from at least serogroups C, W135 and Y (i.e.no MenA saccharide). It is preferred that the protective efficacy ofindividual saccharide antigens is not removed by combining them,although actual immunogenicity (e.g. ELISA titres) may be reduced.

Mixtures of saccharides from more than one serogroup of N.meningitidisare preferred e.g. compositions comprising saccharides from serogroupsA+C, A+W135, A+Y, C+W135, C+Y, W135+Y, A+C+W135, A+C+Y, C+W135+Y,A+C+W135+Y, etc. Preferred compositions comprise saccharides fromserogroups C and Y. Other preferred compositions comprise saccharidesfrom serogroups C, W135 and Y. Compositions in which the capsularsaccharides are from groups A and C only are not preferred (cf. refs.10, 13 & 14).

Where a mixture comprises capsular saccharides from both serogroups Aand C, the ratio (w/w) of MenA saccharide:MenC saccharide may be greaterthan 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or higher).

Where a mixture comprises capsular saccharides from serogroup Y and oneor both of serogroups C and W135, the ratio (w/w) of MenYsaccharide:MenW135 saccharide may be greater than 1 (e.g. 2:1, 3:1, 4:1,5:1, 10:1 or higher) and/or that the ratio (w/w) of MenY saccharide:MenCsaccharide may be less than 1 (e.g. 1:2, 1:3, 1:4, 1:5, or lower).

A typical quantity of each meningococcal saccharide antigen per dose isbetween 1 μg and 20 μg e.g. about 1 μg, about 2.5 μg, about 4 μg, about5 μg, or about 10 μg (expressed as saccharide).

Preferred ratios (w/w) for saccharides from serogroups A:C:W135:Y are:1:1:1:1; 1:1:1:2; 2:1:1:1; 4:2:1:1; 8:4:2:1; 4:2:1:2; 8:4:1:2; 4:2:2:1;2:2:1:1; 4:4:2:1; 2:2:1:2; 4:4:1:2; and 2:2:2:1. Preferred ratios (w/w)for saccharides from serogroups C:W135:Y are: 1:1:1; 1:1:2; 1:1:1;2:1:1; 4:2:1; 2:1:2; 4:1:2; 2:2:1; and 2:1:1. Using a substantiallyequal mass of each saccharide is preferred.

Purification of Capsular Polysaccharides

Meningococcal capsular polysaccharides are typically prepared by aprocess comprising the steps of polysaccharide precipitation (e.g. usinga cationic detergent), ethanol fractionation, cold phenol extraction (toremove protein) and ultracentrifugation (to remove LPS) [e.g. ref. 15].

A more preferred process [7], however, involves polysaccharideprecipitation followed by solubilisation of the precipitatedpolysaccharide using a lower alcohol. Precipitation can be achievedusing a cationic detergent such as tetrabutylammonium andcetyltrimethylammonium salts (e.g. the bromide salts), or hexadimethrinebromide and myristyltrimethylammonium salts. Cetyltrimethylammoniumbromide (‘CTAB’) is particularly preferred [16]. Solubilisation of theprecipitated material can be achieved using a lower alcohol such asmethanol, propan-1-ol, propan-2-ol, butan-1-ol, butan-2-ol,2-methyl-propan-1-ol, 2-methyl-propan-2-ol, diols, etc., but ethanol isparticularly suitable for solubilising CTAB-polysaccharide complexes.Ethanol is preferably added to the precipitated polysaccharide to give afinal ethanol concentration (based on total content of ethanol andwater) of between 50% and 95%.

After re-solubilisation, the polysaccharide may be further treated toremove contaminants. This is particularly important in situations whereeven minor contamination is not acceptable (e.g. for human vaccineproduction). This will typically involve one or more steps of filtratione.g. depth filtration, filtration through activated carbon may be used,size filtration and/or ultrafiltration.

Once filtered to remove contaminants, the polysaccharide may beprecipitated for further treatment and/or processing. This can beconveniently achieved by exchanging cations (e.g. by the addition ofcalcium or sodium salts).

As an alternative to purification, capsular saccharides of the presentinvention may be obtained by total or partial synthesis e.g. Hibsynthesis is disclosed in ref. 17, and MenA synthesis in ref. 18.

The polysaccharide may be chemically modified. For instance, it may bemodified to replace one or more hydroxyl groups with blocking groups.This is particularly useful for reducing hydrolysis for serogroup A [19](see below). De-O-acetylation of saccharides can also be performed.

Serogroup B N.meningitidis

Some compositions of the invention include an antigen from serogroup BN.meningitidis. Unlike serogroups A, C, W135 and Y, the capsularsaccharide of MenB is unsuitable for use as an immunogen in humansbecause of its similarity to self antigens. If a saccharide antigen isto be used for MenB, therefore, it is necessary to use a modifiedsaccharide, such as one in which N-acetyl groups in the saccharide'ssialic acid residues are replaced with N-acyl groups. Suitable N-acylgroups are C₁ to C₈ acyl groups, such as N-propionyl [20]. Rather thanuse a saccharide antigen, however, it is preferred to use a polypeptideantigen.

Thus the composition may include one or more polypeptide antigens whichinduce(s) an immune response that protects against MenB infection and/orthat is bactericidal against MenB. More generally, the composition canpreferably, after administration to a subject, induce an antibodyresponse in that subject that is bactericidal against two or more (e.g.2 or 3) of hypervirulent lineages A4, ET-5 and lineage 3 ofN.meningitidis serogroup B.

The genome sequence of strain MC58 of MenB has been published [21] andsuitable antigens can be selected from the encoded polypeptides [22].Preferred antigens are disclosed in references 22 to 32. Rather thanconsisting of a single antigen, it is preferred that the composition ofthe invention comprises a mixture of 10 or fewer (e.g. 9, 8, 7, 6, 5, 4,3, 2) purified antigens, and it is particularly preferred that thecomposition should not include complex or undefined mixtures of antigense.g. it is preferred not to include outer membrane vesicles (OMVs) inthe composition.

Preferred compositions include one or more of the following fiveantigens [32]: (1) a ‘NadA’ protein, preferably in oligomeric form (e.g.in trimeric form); (2) a ‘741’ protein; (3) a ‘936’ protein; (4) a ‘953’protein; and (5) a ‘287’ protein.

Preferred MenB antigens comprise an amino acid sequence found in one ofstrains are 2996, MC58, 95N477, and 394/98. Protein 287 is preferablyfrom strain 2996 or, more preferably, from strain 394/98. Protein 741 ispreferably from serogroup B strains MC58, 2996, 394/98, or 95N477, orfrom serogroup C strain 90/18311. Strain MC58 is more preferred.Proteins 936, 953 and NadA are preferably from strain 2996. Where acomposition includes a particular protein antigen (e.g. 741 or 287), thecomposition can include that antigen in more than one variant form e.g.the same protein, but from more than one strain. These proteins may beincluded as tandem or separate proteins.

‘NadA’ (Neisserial adhesin A) from MenB is disclosed as protein ‘961’ inreference 25 (SEQ IDs 2943 & 2944) and as ‘NMB1994’ in reference 21 (seealso GenBank accession numbers: 11352904 & 7227256). A detailed study ofthe protein can be found in reference 33. When used according to thepresent invention, NadA may take various forms. Preferred forms of NadAare truncation or deletion variants, such as those disclosed inreferences 29 to 31. In particular, NadA without its C-terminal membraneanchor is preferred (e.g. deletion of residues 351-405 for the 2996strain, to give SEQ ID NO:1 herein), which is sometimes distinguishedherein by the use of a ‘C’ superscript e.g. NadA^((C)). Expression ofNadA without its membrane anchor domain in E. coli results in secretionof the protein into the culture supernatant with concomitant removal ofits 23mer leader peptide (e.g. to leave a 327mer for strain 2996 [SEQ IDNO:2 herein]). Polypeptides without their leader peptides are sometimesdistinguished herein by the use of a ‘NL’ superscript e.g. NadA^((NL))or NadA^((C)(NL))). Preferred NadA polypeptides have an amino acidsequence which: (a) has 50% or more identity (e.g. 60%, 70%, 80%, 90%,95%, 99% or more) to SEQ ID NO:2; and/or (b) comprises a fragment of atleast n consecutive amino acids of SEQ ID NO:1, wherein n is 7 or more(eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,150, 200, 250 or more). Preferred fragments for (b) lack one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the C-terminus and/or the N-terminus of SEQ ID NO:1 (e.g.NadA^((C)), NadA^((NL)), NadA^((C)(NL))). Other preferred fragmentscomprise an epitope from SEQ ID 1, and a particularly preferred fragmentof SEQ ID 1 is SEQ ID 2. These various sequences includes NadA variants(e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.).Various NadA sequences are shown in FIG. 9 of reference 34.

‘741’ protein from MenB is disclosed in reference 25 (SEQ IDs 2535 &2536) and as ‘NMB1870’ in reference 21 (see also GenBank accessionnumber GI:7227128). The corresponding protein in serogroup A [35] hasGenBank accession number 7379322. 741 is naturally a lipoprotein. Whenused according to the present invention, 741 protein may take variousforms. Preferred forms of 741 are truncation or deletion variants, suchas those disclosed in references 29 to 31. In particular, the N-terminusof 741 may be deleted up to and including its poly-glycine sequence(i.e. deletion of residues 1 to 72 for strain MC58 [SEQ ID NO:3herein]), which is sometimes distinguished herein by the use of a ‘AG’prefix. This deletion can enhance expression. The deletion also removes741's lipidation site. Preferred 741 sequences have an amino acidsequence which: (a) has 50% or more identity (e.g. 60%, 70%, 80%, 90%,95%, 99% or more) to SEQ ID NO:3; and/or (b) comprises a fragment of atleast n consecutive amino acids from SEQ ID NO:3, wherein n is 7 or more(eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,150, 200, 250 or more). Preferred fragments for (b) comprise an epitopefrom 741. Other preferred fragments lack one or more amino acids (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminusand/or the N-terminus of SEQ ID NO:3. These sequences include 741variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants,etc.). Various 741 sequences can be found in SEQ IDs 1 to 22 ofreference 31, in SEQ IDs 1 to 23 of reference 36, and in SEQ IDs 1-299of reference 37.

Protein 741 is an extremely effective antigen for elicitinganti-meningococcal antibody responses, and it is expressed across allmeningococcal serogroups. Phylogenetic analysis shows that the proteinsplits into two groups, and that one of these splits again to give threevariants in total [38], and while serum raised against a given variantis bactericidal within the same variant group, it is not active againststrains which express one of the other two variants i.e. there isintra-variant cross-protection, but not inter-variant cross-protection[36,38]. For maximum cross-strain efficacy, therefore, it is preferredthat a composition should include more than one variant of protein 741.An exemplary sequence from each variant is given in SEQ ID NOs: 10, 11and 12 herein, starting with a N-terminal cysteine residue to whichlipid will be covalently attached in the native lipoprotein form. It istherefore preferred that the composition should include at least two of:(1) a first protein, comprising an amino acid sequence having at least a% sequence identity to SEQ ID NO:10 and/or comprising an amino acidsequence consisting of a fragment of at least x contiguous amino acidsfrom SEQ ID NO:10; (2) a second protein, comprising an amino acidsequence having at least b % sequence identity to SEQ ID NO:11 and/orcomprising an amino acid sequence consisting of a fragment of at least ycontiguous amino acids from SEQ ID NO:11; and (3) a third protein,comprising an amino acid sequence having at least c % sequence identityto SEQ ID NO:12 and/or comprising an amino acid sequence consisting of afragment of at least z contiguous amino acids from SEQ ID NO:12. Thevalue of a is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 99.5, or more. The value of b is at least 85 e.g. 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or more. Thevalue of c is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 99.5, or more. The values of a, b and c are notintrinsically related to each other. The value of x is at least 7 e.g.8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160,180, 200, 225, 250). The value of y is at least 7 e.g. 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250).The value of z is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The values of x, yand z are not intrinsically related to each other. It is preferred thatany given 741 amino acid sequence will not fall into more than one ofcategories (1), (2) and (3). Any given 741 sequence will thus fall intoonly one of categories (1), (2) and (3). It is thus preferred that:protein (1) has less than i % sequence identity to protein (2); protein(1) has less than j % sequence identity to protein (3); and protein (2)has less than k % sequence identity to protein (3). The value of i is 60or more (e.g. 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, etc.)and is at most a. The value of j is 60 or more (e.g. 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, etc.) and is at most b. The value of k is 60or more (e.g. 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, etc.)and is at most c. The values of i, j and k are not intrinsically relatedto each other.

‘936’ protein from serogroup B is disclosed in reference 25 (SEQ IDs2883 & 2884) and as ‘NMB2091’ in reference 21 (see also GenBankaccession number GI:7227353). The corresponding gene in serogroup A [35]has GenBank accession number 7379093. When used according to the presentinvention, 936 protein may take various forms. Preferred forms of 936are truncation or deletion variants, such as those disclosed inreferences 29 to 31. In particular, the N-terminus leader peptide of 936may be deleted (e.g. deletion of residues 1 to 23 for strain MC58, togive 936^((NL)) [SEQ ID NO:4 herein]). Preferred 936 sequences have anamino acid sequence which: (a) has 50% or more identity (e.g. 60%, 70%,80%, 90%, 95%, 99% or more) to SEQ ID NO:4; and/or (b) comprises afragment of at least n consecutive amino acids from SEQ ID NO:4, whereinn is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60,70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments for (b)comprise an epitope from 936. Other preferred fragments lack one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the C-terminus and/or the N-terminus of SEQ ID NO:4. Thesesequences include 936 variants (e.g. allelic variants, homologs,orthologs, paralogs, mutants, etc.).

‘953’ protein from serogroup B is disclosed in reference 25 (SEQ IDs2917 & 2918) and as ‘NMB1030’ in reference 21 (see also GenBankaccession number GI:7226269). The corresponding protein in serogroup A[35] has GenBank accession number 7380108. When used according to thepresent invention, 953 protein may take various forms. Preferred formsof 953 are truncation or deletion variants, such as those disclosed inreferences 29 to 31. In particular, the N-terminus leader peptide of 953may be deleted (e.g. deletion of residues 1 to 19 for strain MC58, togive 953^((NL)) [SEQ ID NO:5 herein]. Preferred 953 sequences have anamino acid sequence which: (a) has 50% or more identity (e.g. 60%, 70%,80%, 90%, 95%, 99% or more) to SEQ ID NO:5; and/or (b) comprises afragment of at least n consecutive amino acids from SEQ ID NO:5, whereinn is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60,70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments for (b)comprise an epitope from 953. Other preferred fragments lack one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the C-terminus and/or the N-terminus of SEQ ID NO:5. Thesesequences include 936 variants (e.g. allelic variants, homologs,orthologs, paralogs, mutants, etc.). Allelic forms of 953 can be seen inFIG. 19 of reference 28.

‘287’ protein from serogroup B is disclosed in reference 25 (SEQ IDs3103 & 3104), as ‘NMB2132’ in reference 21, and as ‘GNA2132’ inreference 22 (see also GenBank accession number GI:7227388). Thecorresponding protein in serogroup A [35] has GenBank accession number7379057. When used according to the present invention, 287 protein maytake various forms. Preferred forms of 287 are truncation or deletionvariants, such as those disclosed in references 29 to 31. In particular,the N-terminus of 287 may be deleted up to and including itspoly-glycine sequence (e.g. deletion of residues 1 to 24 for strainMC58, to give AG287 [SEQ ID NO:6 herein]. This deletion can enhanceexpression. Preferred 287 sequences have an amino acid sequence which:(a) has 50% or more identity (e.g. 60%, 70%, 80%, 90%, 95%, 99% or more)to SEQ ID NO:6; and/or (b) comprises a fragment of at least nconsecutive amino acids from SEQ ID NO:6, wherein n is 7 or more (eg. 8,10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments for (b) comprise an epitope from287. Other preferred fragments lack one or more amino acids (e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/orthe N-terminus of SEQ ID NO:6. These sequences include 287 variants(e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.).Allelic forms of 287 can be seen in FIGS. 5 and 15 of reference 28, andin example 13 and FIG. 21 of reference 25 (SEQ IDs 3179 to 3184).

The five basic MenB antigens (NadA, 741, 953, 936 & 287) may be presentin the composition as five separate proteins, but it is preferred thatat least two of the antigens are expressed as a single polypeptide chain(a ‘hybrid’ protein [refs. 29 to 31]) i.e. such that the five antigensform fewer than five polypeptides. Hybrid proteins offer two principaladvantages: first, a protein that may be unstable or poorly expressed onits own can be assisted by adding a suitable hybrid partner thatovercomes the problem; second, commercial manufacture is simplified asonly one expression and purification need be employed in order toproduce two separately-useful proteins. A hybrid protein included in acomposition of the invention may comprise two or more (i.e. 2, 3, 4 or5) of the five basic antigens. Hybrids consisting of two of the fiveantigens are preferred.

Within the combination of five basic antigens, an antigen may be presentin more than one hybrid protein and/or as a non-hybrid protein. It ispreferred, however, that an antigen is present either as a hybrid or asa non-hybrid, but not as both, although it may be useful to includeprotein 741 both as a hybrid and a non-hybrid (preferably lipoprotein)antigen, particularly where more than one variant of 741 is used.

Hybrid proteins can be represented by the formulaNH₂-A-[—X-L-]_(n)—B—COOH, wherein: X is an amino acid sequence of one ofthe five basic antigens; L is an optional linker amino acid sequence; Ais an optional N-terminal amino acid sequence; B is an optionalC-terminal amino acid sequence; and n is 2, 3, 4 or 5.

If a —X— moiety has a leader peptide sequence in its wild-type form,this may be included or omitted in the hybrid protein. In someembodiments, the leader peptides will be deleted except for that of the—X— moiety located at the N-terminus of the hybrid protein i.e. theleader peptide of X₁ will be retained, but the leader peptides of X₂ . .. X_(n) will be omitted. This is equivalent to deleting all leaderpeptides and using the leader peptide of X₁ as moiety -A-.

For each n instances of [—X-L-], linker amino acid sequence -L- may bepresent or absent. For instance, when n=2 the hybrid may beNH₂—X₁-L₁-X₂-L₂-COOH, NH₂—X₁—X₂—COOH, NH₂—X₁-L₁-X₂—COOH,NH₂—X₁—X₂-L₂-COOH, etc. Linker amino acid sequence(s) -L- will typicallybe short (e.g. 20 or fewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples comprise short peptidesequences which facilitate cloning, poly-glycine linkers (i.e.comprising Gly_(n) where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), andhistidine tags (i.e. His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more).Other suitable linker amino acid sequences will be apparent to thoseskilled in the art. A useful linker is GSGGGG (SEQ ID 9), with theGly-Ser dipeptide being formed from a BamHI restriction site, thusaiding cloning and manipulation, and the (Gly)₄ tetrapeptide being atypical poly-glycine linker. If X_(n+1) is a AG protein and L_(n) is aglycine linker, this may be equivalent to X_(n+1) not being a AG proteinand L_(n) being absent.

-A- is an optional N-terminal amino acid sequence. This will typicallybe short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leadersequences to direct protein trafficking, or short peptide sequenceswhich facilitate cloning or purification (e.g. histidine tags i.e.His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitableN-terminal amino acid sequences will be apparent to those skilled in theart. If X₁ lacks its own N-terminus methionine, -A- is preferably anoligopeptide (e.g. with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) whichprovides a N-terminus methionine.

—B— is an optional C-terminal amino acid sequence. This will typicallybe short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples includesequences to direct protein trafficking, short peptide sequences whichfacilitate cloning or purification (e.g. comprising histidine tags i.e.His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences whichenhance protein stability. Other suitable C-terminal amino acidsequences will be apparent to those skilled in the art.

Most preferably, n is 2. Two-antigen hybrids for use in the inventioncomprise: NadA & 741; NadA & 936; NadA & 953; NadA & 287; 741 & 936; 741& 953; 741 & 287; 936 & 953; 936 & 287; 953 & 287. Two preferredproteins are: X₁ is a 936 and X₂ is a 741; X₁ is a 287 and X₂ is a 953.

Two particularly preferred hybrid proteins of the invention are asfollows:

SEQ n A X₁ L₁ X₂ L₂ B ID NO: 2 MA ΔG287 GSGGGG (SEQ ID NO 9) 953^((NL))— — 7 2 M 936^((NL)) GSGGGG (SEQ ID NO 9) ΔG741 — — 8

These two proteins may be used in combination with NadA (particularlywith SEQ ID NO:2). Thus a preferred composition of MenB antigens for usewith the invention thus includes a first polypeptide comprising aminoacid sequence SEQ ID NO:2, a second polypeptide comprising amino acidsequence SEQ ID NO:7 and a third polypeptide comprising amino acidsequence SEQ ID NO:8. This is a preferred group of MenB antigens for usewith the invention.

As mentioned above, compositions of the invention that include MenBantigens can preferably induce a serum bactericidal antibody responsethat is effective against two or three of MenB hypervirulent lineagesA4, ET-5 and lineage 3. They may additionally induce bactericidalantibody responses against one or more of hypervirulent lineagessubgroup I, subgroup III, subgroup Iv-1 or ET-37 complex, and againstother lineages e.g. hyperinvasive lineages. These antibody responses areconveniently measured in mice and are a standard indicator of vaccineefficacy [e.g. see end-note 14 of reference 22]. Serum bactericidalactivity (SBA) measures bacterial killing mediated by complement, andcan be assayed using human or baby rabbit complement. WHO standardsrequire a vaccine to induce at least a 4-fold rise in SBA in more than90% of recipients.

The composition need not induce bactericidal antibodies against each andevery MenB strain within these hypervirulent lineages; rather, for anygiven group of four of more strains of serogroup B meningococcus withina particular hypervirulent lineage, the antibodies induced by thecomposition are bactericidal against at least 50% (e.g. 60%, 70%, 80%,90% or more) of the group. Preferred groups of strains will includestrains isolated in at least four of the following countries: GB, AU,CA, NO, IT, US, NZ, NL, BR, and CU. The serum preferably has abactericidal titre of at least 1024 (e.g. 2¹⁰, 2¹¹, 2¹², 2¹³, 2¹⁴, 2¹⁵,2¹⁶, 2¹⁷, 2¹⁸ or higher, preferably at least 2¹⁴) i.e. the serum is ableto kill at least 50% of test bacteria of a particular strain whendiluted 1/1024, as described in reference 22. Preferred compositions caninduce bactericidal responses against the following strains of serogroupB meningococcus: (i) from cluster A4, strain 961-5945 (B:2b:P1.21,16)and/or strain G2136 (B:-); (ii) from ET-5 complex, strain MC58(B:15:P1.7,16b) and/or strain 44/76 (B:15:P1.7,16); (iii) from lineage3, strain 394/98 (B:4:P1.4) and/or strain BZ198 (B:NT:-). More preferredcompositions can induce bactericidal responses against strains 961-5945,44/76 and 394/98. Strains 961-5945 and G2136 are both Neisseria MLSTreference strains [ids 638 & 1002 in ref. 39]. Strain MC58 is widelyavailable (e.g. ATCC BAA-335) and was the strain sequenced in reference21. Strain 44/76 has been widely used and characterised (e.g. ref. 40)and is one of the Neisseria MLST reference strains [id 237 in ref. 39;row 32 of Table 2 in ref. 41]. Strain 394/98 was originally isolated inNew Zealand in 1998, and there have been several published studies usingthis strain (e.g. refs. 42 & 43). Strain BZ198 is another MLST referencestrain [id 409 in ref. 39; row 41 of Table 2 in ref. 41]. Thecomposition may additionally induce a bactericidal response againstserogroup W135 strain LNP17592 (W135:2a:P1.5,2), from ET-37 complex.This is a Haji strain isolated in France in 2000.

Other MenB polypeptide antigens which may be included in compositions ofthe invention include those comprising one of the following amino acidsequences: SEQ ID NO:650 from ref. 23; SEQ ID NO:878 from ref. 23; SEQID NO:884 from ref. 23; SEQ ID NO:4 from ref. 24; SEQ ID NO:598 fromref. 25; SEQ ID NO:818 from ref. 25; SEQ ID NO:864 from ref. 25; SEQ IDNO:866 from ref. 25; SEQ ID NO:1196 from ref. 25; SEQ ID NO:1272 fromref. 25; SEQ ID NO:1274 from ref. 25; SEQ ID NO:1640 from ref. 25; SEQID NO:1788 from ref. 25; SEQ ID NO:2288 from ref. 25; SEQ ID NO:2466from ref. 25; SEQ ID NO:2554 from ref. 25; SEQ ID NO:2576 from ref. 25;SEQ ID NO:2606 from ref. 25; SEQ ID NO:2608 from ref. 25; SEQ ID NO:2616from ref. 25; SEQ ID NO:2668 from ref. 25; SEQ ID NO:2780 from ref. 25;SEQ ID NO:2932 from ref. 25; SEQ ID NO:2958 from ref. 25; SEQ ID NO:2970from ref. 25; SEQ ID NO:2988 from ref. 25, or a polypeptide comprisingan amino acid sequence which: (a) has 50% or more identity (e.g. 60%,70%, 80%, 90%, 95%, 99% or more) to said sequences; and/or (b) comprisesa fragment of at least n consecutive amino acids from said sequences,wherein n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40,50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments for(b) comprise an epitope from the relevant sequence. More than one (e.g.2, 3, 4, 5, 6) of these polypeptides may be included.

Haemophilus influenzae Type B (Hib)

Where the composition includes a H.influenzae type B antigen, it willtypically be a Hib capsular saccharide antigen. Saccharide antigens fromH.influenzae b are well known.

Advantageously, the Hib saccharide is covalently conjugated to a carrierprotein, in order to enhance its immunogenicity, especially in children.The preparation of polysaccharide conjugates in general, and of the Hibcapsular polysaccharide in particular, is well documented [e.g.references 44 to 52 etc.]. The invention may use any suitable Hibconjugate. Suitable carrier proteins are described below, and preferredcarriers for Hib saccharides are CRM₁₉₇ (‘HbOC’), tetanus toxoid(‘PRP-T’) and the outer membrane complex of N.meningitidis (‘PRP-OMP’).

The saccharide moiety of the conjugate may be a polysaccharide (e.g.full-length polyribosylribitol phosphate (PRP)), but it is preferred tohydrolyse polysaccharides to form oligosaccharides (e.g. MW from ˜1 to˜5 kDa).

A preferred conjugate comprises a Hib oligosaccharide covalently linkedto CRM₁₉₇ via an adipic acid linker [53, 54]. Tetanus toxoid is also apreferred carrier.

Administration of the Hib antigen preferably results in an anti-PRPantibody concentration of ≥0.15 μg/ml, and more preferably ≥1 μg/ml.

Compositions of the invention may comprise more than one Hib antigen.

Where a composition includes a Hib saccharide antigen, it is preferredthat it does not also include an aluminium hydroxide adjuvant. If thecomposition includes an aluminium phosphate adjuvant then the Hibantigen may be adsorbed to the adjuvant [55] or it may be non-adsorbed[12]. Prevention of adsorption can be achieved by selecting the correctpH during antigen/adjuvant mixing, an adjuvant with an appropriate pointof zero charge, and an appropriate order of mixing for the variousdifferent antigens in a composition [56].

Hib antigens may be lyophilised e.g. together with meningococcalantigens.

Streptococcus pneumoniae

Where the composition includes a S.pneumoniae antigen, it will typicallybe a capsular saccharide antigen which is preferably conjugated to acarrier protein [e.g. refs. 57 to 59]. It is preferred to includesaccharides from more than one serotype of S.pneumoniae. For example,mixtures of polysaccharides from 23 different serotype are widely used,as are conjugate vaccines with polysaccharides from between 5 and 11different serotypes [60]. For example, PrevNar™ [61] contains antigensfrom seven serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) with eachsaccharide individually conjugated to CRM₁₉₇ by reductive amination,with 2 μg of each saccharide per 0.5 ml dose (4 μg of serotype 6B), andwith conjugates adsorbed on an aluminium phosphate adjuvant.

Compositions of the invention preferably include at least serotypes 6B,14, 19F and 23F. Conjugates may be adsorbed onto an aluminium phosphate.

As an alternative to using saccharide antigens from pneumococcus, thecomposition may include one or more polypeptide antigens. Genomesequences for several strains of pneumococcus are available [62,63] andcan be subjected to reverse vaccinology [64-67] to identify suitablepolypeptide antigens [68,69]. For example, the composition may includeone or more of the following antigens: PhtA, PhtD, PhtB, PhtE, SpsA,LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp130, as defined inreference 70. The composition may include more than one (e.g. 2, 3, 4,5, 6, 7, 8, 9 10, 11, 12, 13 or 14) of these antigens.

In some embodiments, the composition may include both saccharide andpolypeptide antigens from pneumococcus. These may be used in simpleadmixture, or the pneumococcal saccharide antigen may be conjugated to apneumococcal protein. Suitable carrier proteins for such embodimentsinclude the antigens listed in the previous paragraph [70].

Pneumococcal antigens may be lyophilised e.g. together withmeningococcal and/or Hib antigens.

Modified Serogroup a N.meningitidis Saccharides

Where a composition of the invention includes a MenA saccharide antigen,the antigen is preferably a modified saccharide in which one or more ofthe hydroxyl groups on the native saccharide has/have been replaced by ablocking group [19]. This modification improves resistance tohydrolysis, and means that the serogroup A antigen can be stored andused in a liquid formulation rather than requiring lyophilisation.

The number of monosaccharide units having blocking groups can vary. Forexample, all or substantially all the monosaccharide units may haveblocking groups. Alternatively, at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80% or 90% of the monosaccharide units may have blocking groups. Atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 monosaccharide units mayhave blocking groups.

Likewise, the number of blocking groups on a monosaccharide unit mayvary. For example, the number of blocking groups on a monosaccharideunit may be 1 or 2. The blocking group will generally be at the 4position and/or 3-position of the monosaccharide units.

The terminal monosaccharide unit may or may not have a blocking groupinstead of its native hydroxyl. It is preferred to retain a freeanomeric hydroxyl group on a terminal monosaccharide unit in order toprovide a handle for further reactions (e.g. conjugation). Anomerichydroxyl groups can be converted to amino groups (—NH₂ or —NH-E, where Eis a nitrogen protecting group) by reductive amination (using, forexample, NaBH₃CN/NH₄Cl), and can then be regenerated after otherhydroxyl groups have been converted to blocking groups.

Blocking groups to replace hydroxyl groups may be directly accessiblevia a derivatizing reaction of the hydroxyl group i.e. by replacing thehydrogen atom of the hydroxyl group with another group. Suitablederivatives of hydroxyl groups which act as blocking groups are, forexample, carbamates, sulfonates, carbonates, esters, ethers (e.g. silylethers or alkyl ethers) and acetals. Some specific examples of suchblocking groups are allyl, Aloc, benzyl, BOM, t-butyl, trityl, TBS,TBDPS, TES, TMS, TIPS, PMB, MEM, MOM, MTM, THP, etc. Other blockinggroups that are not directly accessible and which completely replace thehydroxyl group include C₁₋₁₂ alkyl, C₃₋₁₂ alkyl, C₅₋₁₂ aryl, C₅₋₁₂aryl-C₁₋₆ alkyl, NR¹R² (R¹ and R² are defined in the followingparagraph), H, F, Cl, Br, CO₂H, CO₂(C₁₋₆ alkyl), CN, CF₃, CCl₃, etc.Preferred blocking groups are electron-withdrawing groups.

Preferred blocking groups are of the formula: —O—X—Y or —OR³ wherein: Xis C(O), S(O) or SO₂; Y is C₁₋₁₂ alkyl, C₁₋₁₂ alkoxy, C₃₋₁₂ cycloalkyl,C₅₋₁₂ aryl or C₅₋₁₂ aryl-C₁₋₆ alkyl, each of which may optionally besubstituted with 1, 2 or 3 groups independently selected from F, Cl, Br,CO₂H, CO₂(C₁₋₆ alkyl), CN, CF₃ or CCl₃; or Y is NR¹R²; R¹ and R² areindependently selected from H, C₁₋₁₂ alkyl, C₃₋₁₂ cycloalkyl, C₅₋₁₂aryl, C₅₋₁₂ aryl-C₁₋₆ alkyl; or R¹ and R² may be joined to form a C₃₋₁₂saturated heterocyclic group; R³ is C₁₋₁₂ alkyl or C₃₋₁₂ cycloalkyl,each of which may optionally be substituted with 1, 2 or 3 groupsindependently selected from F, Cl, Br, CO₂(C₁₋₆ alkyl), CN, CF₃ or CCl₃;or R³ is C₅₋₁₂ aryl or C₅₋₁₂ aryl-C₁₋₆ alkyl, each of which mayoptionally be substituted with 1, 2, 3, 4 or 5 groups selected from F,Cl, Br, CO₂H, CO₂(C₁₋₆ alkyl), CN, CF₃ or CCl₃. When R³ is C₁₋₁₂ alkylor C₃₋₁₂ cycloalkyl, it is typically substituted with 1, 2 or 3 groupsas defined above. When R¹ and R² are joined to form a C₃₋₁₂ saturatedheterocyclic group, it is meant that R¹ and R² together with thenitrogen atom form a saturated heterocyclic group containing any numberof carbon atoms between 3 and 12 (e.g. C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀,C₁₁, C₁₂). The heterocyclic group may contain 1 or 2 heteroatoms (suchas N, O or S) other than the nitrogen atom. Examples of C₃₋₁₂ saturatedheterocyclic groups are pyrrolidinyl, piperidinyl, morpholinyl,piperazinyl, imidazolidinyl, azetidinyl and aziridinyl.

Blocking groups —O—X—Y and —OR³ can be prepared from —OH groups bystandard derivatizing procedures, such as reaction of the hydroxyl groupwith an acyl halide, alkyl halide, sulfonyl halide, etc. Hence, theoxygen atom in —O—X—Y is preferably the oxygen atom of the hydroxylgroup, while the -X-Y group in —O—X—Y preferably replaces the hydrogenatom of the hydroxyl group.

Alternatively, the blocking groups may be accessible via a substitutionreaction, such as a Mitsonobu-type substitution. These and other methodsof preparing blocking groups from hydroxyl groups are well known.

More preferably, the blocking group is —OC(O)CF3 [71], or a carbamategroup —OC(O)NR¹R², where R¹ and R² are independently selected from C₁₋₆alkyl. More preferably, R¹ and R² are both methyl i.e. the blockinggroup is —OC(O)NMe₂. Carbamate blocking groups have a stabilizing effecton the glycosidic bond and may be prepared under mild conditions.

Preferred modified MenA saccharides contain n monosaccharide units,where at least h % of the monosaccharide units do not have —OH groups atboth of positions 3 and 4. The value of h is 24 or more (e.g. 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98,99 or 100) and is preferably 50 or more. The absent —OH groups arepreferably blocking groups as defined above.

Other preferred modified MenA saccharides comprise monosaccharide units,wherein at least s of the monosaccharide units do not have —OH at the 3position and do not have —OH at the 4 position. The value of s is atleast 1 (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60,70, 80, 90). The absent —OH groups are preferably blocking groups asdefined above.

Suitable modified MenA saccharides for use with the invention have theformula:

wherein

-   -   n is an integer from 1 to 100 (preferably an integer from 15 to        25);    -   T is of the formula (A) or (B):

-   -   each Z group is independently selected from OH or a blocking        group as defined above; and    -   each Q group is independently selected from OH or a blocking        group as defined above;    -   Y is selected from OH or a blocking group as defined above;    -   E is H or a nitrogen protecting group;

and wherein more than about 7% (e.g. 8%, 9%, 10% or more) of the Qgroups are blocking groups.

Each of the n+2 Z groups may be the same or different from each other.Likewise, each of the n+2 Q groups may be the same or different fromeach other. All the Z groups may be OH. Alternatively, at least 10%, 20,30%, 40%, 50% or 60% of the Z groups may be OAc. Preferably, about 70%of the Z groups are OAc, with the remainder of the Z groups being OH orblocking groups as defined above. At least about 7% of Q groups areblocking groups. Preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or even 100% of the Q groups are blocking groups.

Preferred compositions of the invention can be stored for 28 days at 37°C. and, after that period, less than f % of the initial total amount ofconjugated MenA saccharide will be unconjugated, where f is 20, 19, 18,17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or lower.

Oligosaccharides

Capsular saccharides will generally be used in the form ofoligosaccharides. These are conveniently formed by fragmentation ofpurified capsular polysaccharide (e.g. by hydrolysis), which willusually be followed by purification of the fragments of the desiredsize.

Fragmentation of polysaccharides is preferably performed to give a finalaverage degree of polymerisation (DP) in the oligosaccharide of lessthan 30 (e.g. between 10 and 20, preferably around 10 for serogroup A;between 15 and 25 for serogroups W135 and Y, preferably around 15-20;between 12 and 22 for serogroup C; etc.). DP can conveniently bemeasured by ion exchange chromatography or by colorimetric assays [72].

Preferred MenC saccharide antigens are disclosed in reference 10, asused in Menjugate™.

If hydrolysis is performed, the hydrolysate will generally be sized inorder to remove short-length oligosaccharides [73]. This can be achievedin various ways, such as ultrafiltration followed by ion-exchangechromatography. Oligosaccharides with a degree of polymerisation of lessthan or equal to about 6 are preferably removed for serogroup A, andthose less than around 4 are preferably removed for serogroups W135 andY.

Covalent Conjugation

Capsular saccharides in compositions of the invention will usually beconjugated to carrier protein(s). In general, conjugation enhances theimmunogenicity of saccharides as it converts them from T-independentantigens to T-dependent antigens, thus allowing priming forimmunological memory. Conjugation is particularly useful for paediatricvaccines [e.g. ref. 74] and is a well known technique [e.g. reviewed inrefs. 44 to 52, etc.].

Preferred carrier proteins are bacterial toxins or toxoids, such asdiphtheria toxoid or tetanus toxoid. The CRM₁₉₇ diphtheria toxoid[75-77] is particularly preferred. Other suitable carrier proteinsinclude the N.meningitidis outer membrane protein [78], syntheticpeptides [79,80], heat shock proteins [81,82], pertussis proteins [83,84], cytokines [85], lymphokines [85], hormones [85], growth factors[85], artificial proteins comprising multiple human CD4⁺ T cell epitopesfrom various pathogen-derived antigens [86], protein D from H.influenzae[87,88], pneumococcal surface protein PspA [89], iron-uptake proteins[90], toxin A or B from C.difficile [91], etc. Preferred carriers arediphtheria toxoid, tetanus toxoid, H.influenzae protein D, and CRM₁₉₇.

Within a composition of the invention, it is possible to use more thanone carrier protein e.g. to reduce the risk of carrier suppression. Thusdifferent carrier proteins can be used for different serogroups e.g.serogroup A saccharides might be conjugated to CRM₁₉₇ while serogroup Csaccharides might be conjugated to tetanus toxoid. It is also possibleto use more than one carrier protein for a particular saccharide antigene.g. serogroup A saccharides might be in two groups, with someconjugated to CRM₁₉₇ and others conjugated to tetanus toxoid. Ingeneral, however, it is preferred to use the same carrier protein forall saccharides.

A single carrier protein might carry more than one saccharide antigen[92]. For example, a single carrier protein might have conjugated to itsaccharides from serogroups A and C. To achieve this goal, saccharidescan be mixed prior to the conjugation reaction. In general, however, itis preferred to have separate conjugates for each serogroup.

Conjugates with a saccharide:protein ratio (w/w) of between 1:5 (i.e.excess protein) and 5:1 (i.e. excess saccharide) are preferred. Ratiosbetween 1:2 and 5:1 are preferred, as are ratios between 1:1.25 and1:2.5 are more preferred. Excess carrier protein may be preferred forMenA and MenC.

Conjugates may be used in conjunction with free carrier protein [93].When a given carrier protein is present in both free and conjugated formin a composition of the invention, the unconjugated form is preferablyno more than 5% of the total amount of the carrier protein in thecomposition as a whole, and more preferably present at less than 2% byweight.

Any suitable conjugation reaction can be used, with any suitable linkerwhere necessary.

The saccharide will typically be activated or functionalised prior toconjugation. Activation may involve, for example, cyanylating reagentssuch as CDAP (e.g. 1-cyano-4-dimethylamino pyridinium tetrafluoroborate[94,95,etc.]). Other suitable techniques use carbodiimides, hydrazides,active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide,S—NHS, EDC, TSTU; see also the introduction to reference 50).

Linkages via a linker group may be made using any known procedure, forexample, the procedures described in references 96 and 97. One type oflinkage involves reductive amination of the polysaccharide, coupling theresulting amino group with one end of an adipic acid linker group, andthen coupling a protein to the other end of the adipic acid linker group[48, 98, 99]. Other linkers include B-propionamido [100],nitrophenyl-ethylamine [101], haloacyl halides [102], glycosidiclinkages [103], 6-aminocaproic acid [104], ADH [105], C₄ to C₁₂ moieties[106] etc. As an alternative to using a linker, direct linkage can beused. Direct linkages to the protein may comprise oxidation of thepolysaccharide followed by reductive amination with the protein, asdescribed in, for example, references 107 and 108.

A process involving the introduction of amino groups into the saccharide(e.g. by replacing terminal ═O groups with —NH₂) followed byderivatisation with an adipic diester (e.g. adipic acidN-hydroxysuccinimido diester) and reaction with carrier protein ispreferred. Another preferred reaction uses CDAP activation with aprotein D carrier e.g. for MenA or MenC.

After conjugation, free and conjugated saccharides can be separated.There are many suitable methods, including hydrophobic chromatography,tangential ultrafiltration, diafiltration etc. [see also refs. 109 &110, etc.].

Where the composition of the invention includes a conjugatedoligosaccharide, it is preferred that oligosaccharide preparationprecedes conjugation.

Preparation of Compositions of the Invention

Compositions of the invention comprise capsular saccharides from atleast two of serogroups A, C, W135 and Y of N.meningitidis. Thesaccharides are preferably prepared separately (including anyfragmentation, conjugation, etc.) and then admixed to give a compositionof the invention.

Where the composition comprises capsular saccharide from serogroup A,however, it is preferred that the serogroup A saccharide is not combinedwith the other saccharide(s) until shortly before use, in order tominimise the potential for hydrolysis. This can conveniently be achievedby having the serogroup A component (typically together with appropriateexcipients) in lyophilised form and the other serogroup component(s) inliquid form (also with appropriate excipients), with the liquidcomponents being used to reconstitute the lyophilised MenA componentwhen ready for use. Where an aluminium salt adjuvant is used, it ispreferred to include the adjuvant in the vial containing the with theliquid vaccine, and to lyophilise the MenA component without adjuvant.

A composition of the invention may thus be prepared from a kitcomprising: (a) capsular saccharide from N.meningitidis serogroup A, inlyophilised form; and (b) capsular saccharide(s) from one or more (e.g.1, 2, 3) of N.meningitidis serogroups C, W135 and Y, in liquid form. Theinvention also provides a method for preparing a composition of theinvention, comprising mixing a lyophilised capsular saccharide fromN.meningitidis serogroup A with capsular saccharide(s) from one or more(e.g. 1, 2, 3) of N.meningitidis serogroups C, W135 and Y, wherein saidone or more saccharides are in liquid form.

The invention also provides a composition of the invention, comprisingcapsular saccharide(s) from N.meningitidis serogroups C, W135 and Y,wherein saccharides are in liquid form. This composition may be packagedwith a lyophilised serogroup A saccharide antigen, for reconstitution,or it may be used as a composition on its own e.g. where immunisationagainst serogroup A is not desired.

The invention also provides a kit comprising: (a) a first containercontaining capsular saccharides from two or more of N.meningitidisserogroups C, W135 and Y, all in lyophilised form; and (b) a secondcontainer containing, in liquid form, (i) capsular saccharides from noneor one of N.meningitidis serogroups C, W135 and Y, and optionally (ii)further antigens (see below) that do not include meningococcal capsularsaccharides, wherein, reconstitution of the contents of container (a) bythe contents of container (b) provides a composition of the invention.

Presentation of Compositions of the Invention

Compositions of the invention may be presented and packaged in variousways.

The compositions may be presented in vials, or they may be presented inready-filled syringes. The syringes may be supplied with or withoutneedles. A syringe will include a single dose of the composition,whereas a vial may include a single dose or multiple doses. Injectablecompositions will usually be liquid solutions or suspensions.Alternatively, they may be presented in solid form (e.g. freeze-dried)for solution or suspension in liquid vehicles prior to injection.

Compositions of the invention may be packaged in unit dose form or inmultiple dose form. For multiple dose forms, vials are preferred topre-filled syringes. Effective dosage volumes can be routinelyestablished, but a typical human dose of the composition for injectionhas a volume of 0.5 ml.

Where a composition of the invention is to be prepared extemporaneouslyprior to use (e.g. where serogroup A saccharide is presented inlyophilised form) and is presented as a kit, the kit may comprise twovials, or it may comprise one ready-filled syringe and one vial, withthe contents of the syringe being used to reactivate the contents of thevial prior to injection.

Within each dose, the amount of an individual saccharide antigen willgenerally be between 1-50 μg (measured as mass of saccharide), withabout 2.5 μg, 5 μg or 10 μg of each being preferred. With A:C:W135:Yweight ratios of 1:1:1:1; 1:1:1:2; 2:1:1:1; 4:2:1:1; 8:4:2:1; 4:2:1:2;8:4:1:2; 4:2:2:1; 2:2:1:1; 4:4:2:1; 2:2:1:2; 4:4:1:2; and 2:2:2:1,therefore, the amount represented by the FIG. 1 is preferably about 2.5μg, 5 μg or 10 μg. For a 1:1:1:1 ratio A:C:W:Y composition and a 10 μgper saccharide, therefore, 40 μg saccharide is administered per dose.Preferred compositions have about the following μg saccharide per dose:

A 10 0 0 0 10 5 2.5 C 10 10 5 2.5 5 5 2.5 W135 10 10 5 2.5 5 5 2.5 Y 1010 5 2.5 5 5 2.5

Preferred compositions of the invention comprise less than 50 μgmeningococcal saccharide per dose. Other preferred compositions comprise≤40 μg meningococcal saccharide per dose. Other preferred compositionscomprise ≤30 μg meningococcal saccharide per dose. Other preferredcompositions comprise ≤25 μg meningococcal saccharide per dose. Otherpreferred compositions comprise ≤20 μg meningococcal saccharide perdose. Other preferred compositions comprise ≤10 μg meningococcalsaccharide per dose but, ideally, compositions of the invention compriseat least 10 μg meningococcal saccharide per dose.

Compositions of the invention are preferably sterile. They arepreferably pyrogen-free. They are preferably buffered e.g. at between pH6 and pH 8, generally around pH 7. Where a composition comprises analuminium hydroxide salt, it is preferred to use a histidine buffer[111]. Compositions of the invention may be isotonic with respect tohumans.

Adjuvants

The compositions will generally include one or more adjuvants. Theadjuvant(s) may be added to saccharides before and/or after they areadmixed to form a composition of the invention, but it is preferred tocombine adjuvant with a saccharide antigen prior to admixing ofdifferent saccharides.

However, it is not necessary that each saccharide must be adjuvantedprior to such admixing. Excess adjuvant can be included in onesaccharide preparation such that, when further unadjuvanted saccharideantigen(s) is/are added, the excess is diluted to a desired finalconcentration. In one particular embodiment, where the composition ofthe invention is prepared from a lyophilised antigen (e.g. a lyophilisedserogroup A component) it may be preferred not to include an adjuvant inthe lyophilised material.

Preferred adjuvants for inclusion in compositions of the invention arealuminium salts (alum), such as aluminium hydroxides (includingoxyhydroxides), aluminium phosphates (including hydroxyphosphates),aluminium sulfate, etc [Chapters 8 & 9 in ref. 112]. Aluminiumhydroxyphosphate is particularly preferred, particularly in compositionswhich include a H.influenzae saccharide antigen, and a typical adjuvantis amorphous aluminium hydroxyphosphate with PO₄/Al molar ratio between0.84 and 0.92, included at 0.6 mg Al³⁺/ml. Adsorption with a low dose ofaluminium phosphate may be used e.g. between 50 and 100 μg Al³⁺ perconjugate per dose. Where there is more than one conjugate in acomposition, not all conjugates need to be adsorbed. The Menjugate™ andNeisVac™ MenC conjugates use a hydroxide adjuvant, whereas Meningitec™uses a phosphate.

Calcium phosphate is another preferred adjuvant.

Other adjuvants which may be used in addition to or in place ofaluminium salts include:

A. Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulphates, etc. [e.g. see chapters 8 & 9 of ref. 112], or mixtures ofdifferent mineral compounds, with the compounds taking any suitable form(e.g. gel, crystalline, amorphous, etc.), and with adsorption beingpreferred. The mineral containing compositions may also be formulated asa particle of metal salt [113].

B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59™ [Chapter 10 of ref. 112;see also ref. 114] (5% Squalene, 0.5% TWEEN80™ and 0.5% SPAN 85™,formulated into submicron particles using a microfluidizer). CompleteFreund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may alsobe used.

C. Saponin Formulations [Chapter 22 of Ref 112]

Saponin formulations may also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponin from the bark of theQuillaia saponaria Molina tree have been widely studied as adjuvants.Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs. QS21 is marketed as Stimulon™.

Saponin compositions have been purified using HPLC and RP-HPLC. Specificpurified fractions using these techniques have been identified,including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, thesaponin is QS21. A method of production of QS21 is disclosed in ref.114. Saponin formulations may also comprise a sterol, such ascholesterol [115].

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexes (ISCOMs) [chapter 23 ofref. 112]. ISCOMs typically also include a phospholipid such asphosphatidylethanolamine or phosphatidylcholine. Any known saponin canbe used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA,QHA and QHC. ISCOMs are further described in refs. 115-117. Optionally,the ISCOMS may be devoid of additional detergent [118].

A review of the development of saponin based adjuvants can be found inrefs. 119 & 120.

D. Virosomes and Virus-Like Particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,QB-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein p1). VLPs are discussed furtherin refs. 121-126. Virosomes are discussed further in, for example, ref.127

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacteriallipopolysaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred“small particle” form of 3 De-O-acylated monophosphoryl lipid A isdisclosed in ref. 128. Such “small particles” of 3dMPL are small enoughto be sterile filtered through a 0.22 μm membrane [128]. Other non-toxicLPS derivatives include monophosphoryl lipid A mimics, such asaminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [129,130].

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in refs. 131 & 132.

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (adinucleotide sequence containing an unmethylated cytosine linked by aphosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. References 133, 134 and 135 disclose possible analogsubstitutions e.g. replacement of guanosine with2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotidesis further discussed in refs. 136-141.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT [142]. The CpG sequence may be specific for inducing a Thl immuneresponse, such as a CpG-A ODN, or it may be more specific for inducing aB cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed inrefs. 143-145. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, refs. 142 & 146-148.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (E. coli heat labile enterotoxin “LT”), cholera(“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylatingtoxins as mucosal adjuvants is described in ref. 149 and as parenteraladjuvants in ref. 150. The toxin or toxoid is preferably in the form ofa holotoxin, comprising both A and B subunits. Preferably, the A subunitcontains a detoxifying mutation; preferably the B subunit is notmutated. Preferably, the adjuvant is a detoxified LT mutant such asLT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins anddetoxified derivatives thereof, particularly LT-K63 and LT-R72, asadjuvants can be found in refs. 151-158. Numerical reference for aminoacid substitutions is preferably based on the alignments of the A and Bsubunits of ADP-ribosylating toxins set forth in ref. 159, specificallyincorporated herein by reference in its entirety.

F. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12 [160], etc.) [161], interferons (e.g. interferon-y),macrophage colony stimulating factor, and tumor necrosis factor.

G. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres [162] or mucoadhesives such as cross-linked derivatives ofpoly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention [163].

H. Microparticles Microparticles may also be used as adjuvants in theinvention. Microparticles (i.e. a particle of ˜100 nm to ˜150 μm indiameter, more preferably ˜200 nm to ˜30 μm in diameter, and mostpreferably ˜500 nm to ˜10 μm in diameter) formed from materials that arebiodegradable and non-toxic (e.g. a poly(a-hydroxy acid), apolyhydroxybutyric acid, a polyorthoester, a polyanhydride, apolycaprolactone, etc.), with poly(lactide-co-glycolide) are preferred,optionally treated to have a negatively-charged surface (e.g. with SDS)or a positively-charged surface (e.g. with a cationic detergent, such asCTAB).

I. Liposomes (Chapters 13 & 14 of Ref 112)

Examples of liposome formulations suitable for use as adjuvants aredescribed in refs. 164-166.

J. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters [167]. Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol [168] as well as polyoxyethylene alkyl ethers or estersurfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol [169]. Preferred polyoxyethylene ethersare selected from the following group: polyoxyethylene-9-lauryl ether(laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steorylether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether,and polyoxyethylene-23-lauryl ether.

K. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in refs. 170 and 171.

L. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

M. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in theinvention include Imiquamod and its homologues (e.g. “Resiquimod 3M”),described further in refs. 172 and 173.

The invention may also comprise combinations one or more of theadjuvants identified above. For example, the following adjuvantcompositions may be used in the invention: (1) a saponin and anoil-in-water emulsion [175]; (2) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL) [176]; (3) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL)+a cholesterol; (4) a saponin (e.g.QS21)+3dMPL+IL-12 (optionally+a sterol) [177]; (5) combinations of 3dMPLwith, for example, QS21 and/or oil-in-water emulsions [178]; (6) SAF,containing 10% squalane, 0.4% TWEEN 80™, 5% pluronic-block polymer L121,and thr-MDP, either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion. (7) RIBI™) adjuvant system(RAS), (Ribi Immunochem) containing 2% squalene, 0.2% TWEEN 80™, and oneor more bacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (DETOX™); and (8) one or more mineralsalts (such as an aluminum salt)+a non-toxic derivative of LPS (such as3dMPL).Other substances that act as immunostimulating agents aredisclosed in chapter 7 of ref. 112.

Where an aluminium phosphate it used, it is possible to adsorb one ormore of the saccharides to the aluminium salt, but it is preferred notto do so, and this is favoured by including free phosphate ions insolution (e.g. by the use of a phosphate buffer). Where an aluminiumhydroxide is used, it is preferred to adsorb the saccharides to thesalt. The use of aluminium hydroxide as an adjuvant may be preferred forsaccharide from serogroup A.

It is possible in compositions of the invention to adsorb some antigensto an aluminium hydroxide but to have other antigens in association withan aluminium phosphate. For tetravalent N.meningitidis serogroupcombinations, for example, the following permutations are available:

Serogroup Aluminium salt (H = a hydroxide; P = a phosphate) A P H P H HH P P P H H H P P P H C P H H P H H P H H P P H P H P P W135 P H H H P HH P H H P P P P H P Y P H H H H P H H P P H P H P P P

For trivalent N.meningitidis serogroup combinations, the followingpermutations are available:

Serogroup Aluminium salt (H = a hydroxide; P = a phosphate) C P H H H PP P H W135 P H H P H P H P Y P H P H H H P P

Further Components of the Compositions

In addition to antigens described above, compositions of the inventionmay include meningococcal protein antigens.

Non-meningococcal and non-neisserial antigens, preferably ones that donot diminish the immune response against the meningococcal components,may also be included. Ref. 174, for instance, discloses combinations ofoligosaccharides from N.meningitidis serogroups B and C together withthe Hib saccharide. Antigens from pneumococcus, hepatitis A virus,hepatitis B virus, B.pertussis, diphtheria, tetanus, polio and/orH.influenzae are preferred. Particularly preferred antigens include:

-   -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter        3 of ref. 175].    -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of        ref. 175].    -   pertussis holotoxin (PT) and filamentous haemagglutinin (FHA)        from B.pertussis, optionally also in combination with pertactin        and/or agglutinogens 2 and 3 [e.g. refs. 176 & 177].    -   cellular pertussis antigen.    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. 178, 179].    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. 179, 180], with surface antigen preferably        being adsorbed onto an aluminium phosphate [181].    -   Preparations of N.meningitidis serogroup B microvesicles [182],        ‘native OMVs’ [183], blebs or outer membrane vesicles [e.g.        refs. 184 to 185 186 187 188 189 etc.]. These may be prepared        from bacteria which have been genetically manipulated [190-191        192 193] e.g. to increase immunogenicity (e.g. hyper-express        immunogens), to reduce toxicity, to inhibit capsular        polysaccharide synthesis, to down-regulate PorA expression, etc.        They may be prepared from hyperblebbing strains [194-195 196        197]. Vesicles from a non-pathogenic Neisseria may be included        [198]. OMVs may be prepared without the use of detergents        [199,200]. They may express non-Neisserial proteins on their        surface [201]. They may be LPS-depleted. They may be mixed with        recombinant antigens [184,202]. Vesicles from bacteria with        different class I outer membrane protein subtypes may be used        e.g. six different subtypes [203,204] using two different        genetically-engineered vesicle populations each displaying three        subtypes, or nine different subtypes using three different        genetically-engineered vesicle populations each displaying three        subtypes, etc. Useful subtypes include: P1.7,16; P1.5-1,2-2;        P1.19,15-1; P1.5-2,10; P1.12-1,13; P1.7-2,4; P1.22,14; P1.7-1,1;        P1.18-1,3,6.    -   polio antigen(s) [e.g. 205, 206] such as IPV.

The mixture may comprise one or more of these further antigens, whichmay be detoxified where necessary (e.g. detoxification of pertussistoxin by chemical and/or genetic means).

Where a diphtheria antigen is included in the mixture it is preferredalso to include tetanus antigen and pertussis antigens. Similarly, wherea tetanus antigen is included it is preferred also to include diphtheriaand pertussis antigens. Similarly, where a pertussis antigen is includedit is preferred also to include diphtheria and tetanus antigens. SuchDTP combinations can be used to reconstitute lyophilised conjugates.

Antigens in the mixture will typically be present at a concentration ofat least 1 μg/ml each. In general, the concentration of any givenantigen will be sufficient to elicit an immune response against thatantigen.

As an alternative to using proteins antigens in the mixture, nucleicacid encoding the antigen may be used. Protein components of the mixturemay thus be replaced by nucleic acid (preferably DNA e.g. in the form ofa plasmid) that encodes the protein. Similarly, compositions of theinvention may comprise proteins which mimic saccharide antigens e.g.mimotopes [207] or anti-idiotype antibodies. These may replaceindividual saccharine components, or may supplement them. As an example,the vaccine may comprise a peptide mimic of the MenC [208] or the MenA[209] capsular polysaccharide in place of the saccharide itself.

Compositions of the invention may include an antimicrobial, particularlywhen packaged in multiple dose format.

Compositions of the invention may comprise detergent (e.g. a TWEEN™(polysorbate), such as TWEEN80™) at low levels (e.g. <0.01%).

Compositions of the invention may include sodium salts (e.g. sodiumchloride) to give tonicity. A concentration of 10±2 mg/ml NaCl istypical.

Compositions of the invention will generally include a buffer. Aphosphate buffer is typical.

Compositions of the invention may comprise a sugar alcohol (e.g.mannitol) or a disaccharide (e.g. sucrose [210] or trehalose [211]) e.g.at around 15-30 mg/ml (e.g. 25 mg/ml), particularly if they are to belyophilised or if they include material which has been reconstitutedfrom lyophilised material. The pH of a composition for lyophilisationmay be adjusted to around 6.1 prior to lyophilisation.

The invention provides a composition comprising conjugated capsularsaccharides from at least three of serogroups A, C, W135 and Y ofN.meningitidis, wherein the composition comprises sucrose. Thesaccharides are preferably oligosaccharides. The composition may contain≤50 μg total meningococcal saccharide per dose (e.g. ≤40 μg, ≤30 μg, ≤20μg, ≤10 μg). Preferred compositions include: serogroups A, C, W135;serogroups A, C, Y; serogroups C, W135, Y; and all four of serogroups A,C, W135 and Y. Modified MenA saccharide may be used. The composition maybe in aqueous or dried (e.g. lyophilised) form. When in aqueous form,the concentration of sucrose is preferably between 5-50 mg/ml e.g. about25 mg/ml. When in lyophilised form, it is preferred that the compositiondoes not include an aluminium salt adjuvant. The composition mayadditionally comprise an antigen from one or more of: (a) serogroup BN.meningitidis; (b) Haemophilus influenzae type B; and/or (c)Streptococcus pneumoniae.

Immunogenicity

Compositions of the invention are immunogenic. Preferred immunogeniccompositions are vaccines. Vaccines according to the invention mayeither be prophylactic (i.e. to prevent infection) or therapeutic (i.e.to treat disease after infection), but will typically be prophylactic.

Immunogenic compositions and vaccines of the invention will, in additionto the antigens described above, typically comprise ‘pharmaceuticallyacceptable carriers’, which include any carrier that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolised macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,sucrose, trehalose [211], lactose, lipid aggregates (such as oildroplets or liposomes), and inactive virus particles. Such carriers arewell known to those of ordinary skill in the art. The vaccines may alsocontain diluents, such as water, saline, glycerol, etc. Additionally,auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, may be present. Sterilepyrogen-free, phosphate-buffered physiologic saline is a typicalcarrier. A thorough discussion of pharmaceutically acceptable excipientsis available in ref. 212.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of each antigen, as well as any other of theabove-mentioned components, as needed. By ‘immunologically effectiveamount’, it is meant that the administration of that amount to anindividual, either in a single dose or as part of a series, is effectivefor treatment or prevention. This amount varies depending upon thehealth and physical condition of the individual to be treated, age, thetaxonomic group to be treated (e.g. non-human primate, human, etc.), thecapacity of the individual's immune system to synthesise antibodies, thedegree of protection desired, the formulation of the vaccine, thetreating doctor's assessment of the medical situation, and otherrelevant factors. The amount falls in a relatively broad range that canbe determined through routine trials, and a typical quantity of eachmeningococcal saccharide antigen per dose is between 1 μg and 20 μg e.g.about 1 μg, about 2.5 μg, about 4 μg, about 5 μg, or about 10 μg(expressed as saccharide).

Immunogenicity of compositions of the invention can be determined byadministering them to test subjects (e.g. children 12-16 months age, oranimal models [213]) and then determining standard parameters includingserum bactericidal antibodies (SBA) and ELISA titres (GMT) of total andhigh-avidity anti-capsule IgG. These immune responses will generally bedetermined around 4 weeks after administration of the composition, andcompared to values determined before administration of the composition.A SBA increase of at least 4-fold or 8-fold is preferred. Where morethan one dose of the composition is administered, more than onepost-administration determination may be made.

Preferred compositions of the invention can confer an antibody titre ina patient that is superior to the criterion for seroprotection for eachantigenic component for an acceptable percentage of human subjects.Antigens with an associated antibody titre above which a host isconsidered to be seroconverted against the antigen are well known, andsuch titres are published by organisations such as WHO. Preferably morethan 80% of a statistically significant sample of subjects isseroconverted, more preferably more than 90%, still more preferably morethan 93% and most preferably 96-100%.

Administration of Compositions of the Invention

Compositions of the invention are injectable.

Parenteral injection may be subcutaneous, intraperitoneal, intravenousor intramuscular. Intramuscular administration to the thigh or the upperarm is preferred. Injection may be via a needle (e.g. a hypodermicneedle), but needle-free injection may alternatively be used. A typicalintramuscular dose value is 0.5 ml.

Administration may be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule. A primary dose schedule maybe followed by a booster dose schedule. Suitable timing between primingdoses (e.g. between 4-16 weeks), and between priming and boosting, canbe routinely determined.

Administration will generally be to an animal and, in particular, humansubjects can be treated. The compositions are particularly useful forvaccinating children and teenagers.

Medical Methods and Uses

The invention provides a method of raising an immune response in apatient, comprising injecting a patient with a composition of theinvention. The immune response is preferably protective againstmeningococcal disease, and may comprise a humoral immune response and/ora cellular immune response. The patient is preferably a child.

The method may raise a booster response, in a patient that has alreadybeen primed against N.meningitidis.

The invention also provides the use of (i) capsular saccharides from atleast two of serogroups A, C, W135 and Y of N.meningitidis wherein saidcapsular saccharides are conjugated to carrier protein(s) and/or areoligosaccharides, and (ii) an antigen from one or more of: (a) serogroupB N.meningitidis; (b) Haemophilus influenzae type B; and/or (c)Streptococcus pneumoniae, in the manufacture of a injectable medicamentfor raising an immune response in an animal. The medicament ispreferably for the prevention and/or treatment of bacterial meningitis.

One way of checking efficacy of therapeutic treatment involvesmonitoring bacterial infection after administration of the compositionof the invention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses against the administered antigensafter administration of the composition.

Heterologous Host

Whilst expression of polypeptides for use in compositions of theinvention may take place in the native host (e.g. in a N.meningitidis orS.pneumoniae), a heterologous host is preferably used. The heterologoushost may be prokaryotic (e.g. a bacterium) or eukaryotic. It ispreferably E. coli, but other suitable hosts include Bacillus subtilis,Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisserialactamica, Neisseria cinerea, Mycobacteria (e.g. M. tuberculosis),yeast, etc.

General

The term “comprising” means “including” as well as “consisting” e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

Bacterial strains may be indicated as a subscript e.g. 741_(MC58) isprotein 741 from strain MC58. Unless otherwise stated, proteinsmentioned herein (e.g. with no subscript) are from N.meningitidis strain2996, which can be taken as a ‘reference’ strain. It will beappreciated, however, that the invention is not in general limited bystrain. As mentioned above, general references to a protein (e.g. ‘287’,‘919’ etc.) may be taken to include that protein from any strain. Thiswill typically have sequence identity to 2996 of 90% or more (eg. 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). Where hybrid proteinsare used, the individual antigens within the hybrid (i.e. individual —X—moieties) may be from one or more strains. Where n=2, for instance, X₂may be from the same strain as X₁ or from a different strain. Where n=3,the strains might be (i) X₁=X₂=X₃ (ii) X₁=X₂A3 (iii) X₁≠X₂=X₃ (iv)X₁≠X₂≠X₃ or (v) X₁=X₃≠X₂, etc.

The term “alkyl” refers to alkyl groups in both straight and branchedforms, The alkyl group may be interrupted with 1, 2 or 3 heteroatomsselected from —O—, —NH— or —S—. The alkyl group may also be interruptedwith 1, 2 or 3 double and/or triple bonds. However, the term “alkyl”usually refers to alkyl groups having no heteroatom interruptions ordouble or triple bond interruptions. Where reference is made to C₁₋₁₂alkyl, it is meant the alkyl group may contain any number of carbonatoms between 1 and 12 (e.g. C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀,C₁₁, C₁₂). Similarly, where reference is made to C₁₋₆ alkyl, it is meantthe alkyl group may contain any number of carbon atoms between 1 and 6(e.g. C₁, C₂, C₃, C₄, C₅, C₆).

The term “cycloalkyl” includes cycloalkyl, polycycloalkyl, andcycloalkenyl groups, as well as combinations of these with alkyl groups,such as cycloalkylalkyl groups. The cycloalkyl group may be interruptedwith 1, 2 or 3 heteroatoms selected from —O—, —NH— or —S—. However, theterm “cycloalkyl” usually refers to cycloalkyl groups having noheteroatom interruptions Examples of cycloalkyl groups includecyclopentyl, cyclohexyl, cyclohexenyl, cyclohexylmethyl and adamantylgroups. Where reference is made to C₃₋₁₂ cycloalkyl, it is meant thatthe cycloalkyl group may contain any number of carbon atoms between 3and 12 (e.g. C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂).

The term “aryl” refers to an aromatic group, such as phenyl or naphthyl.Where reference is made to C₅₋₁₂ aryl, it is meant that the aryl groupmay contain any number of carbon atoms between 5 and 12 (e.g. C₅, C₆,C₇, C₈, C₉, C₁₀, C₁₁, C₁₂).

The term “C₅₋₁₂ aryl-C₁₋₆ alkyl” refers to groups such as benzyl,phenylethyl and naphthylmethyl.

Nitrogen protecting groups include silyl groups (such as TMS, TES, TBS,TIPS), acyl derivatives (such as phthalimides, trifluoroacetamides,methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl (Boc),benzyloxycarbonyl (Z or Cbz), 9-fluorenylmethoxycarbonyl (Fmoc),2-(trimethylsilyl)ethoxy carbonyl, 2,2,2-trichloroethoxycarbonyl(Troc)), sulfonyl derivatives (such as β-trimethylsilylethanesulfonyl(SES)), sulfenyl derivatives, C₁₋₁₂ alkyl, benzyl, benzhydryl, trityl,9-phenylfluorenyl etc. A preferred nitrogen protecting group is Fmoc.

It will be appreciated that sugar rings can exist in open and closedform and that, whilst closed forms are shown in structural formulaeherein, open forms are also encompassed by the invention.

Sequences included to facilitate cloning or purification, etc., do notnecessarily contribute to the invention and may be omitted or removed.

Polypeptides of the invention can be prepared by various means (e.g.recombinant expression, purification from cell culture, chemicalsynthesis (at least in part), etc.) and in various forms (e.g. native,fusions, non-glycosylated, lipidated, etc.). They are preferablyprepared in substantially pure form (i.e. substantially free from otherN.meningitidis or host cell proteins).

Nucleic acid according to the invention can be prepared in many ways(e.g. by chemical synthesis (at least in part), from genomic or cDNAlibraries, from the organism itself, etc.) and can take various forms(e.g. single stranded, double stranded, vectors, probes, etc.). They arepreferably prepared in substantially pure form (i.e. substantially freefrom other N.meningitidis or host cell nucleic acids). The term “nucleicacid” includes DNA and RNA, and also their analogues, such as thosecontaining modified backbones (e.g. phosphorothioates, etc.), and alsopeptide nucleic acids (PNA) etc. The invention includes nucleic acidcomprising sequences complementary to those described above (eg. forantisense or probing purposes).

After serogroup, meningococcal classification includes serotype,serosubtype and then immunotype, and the standard nomenclature listsserogroup, serotype, serosubtype, and immunotype, each separated by acolon e.g. B:4:P1.15:L3,7,9. Within serogroup B, some lineages causedisease often (hyperinvasive), some lineages cause more severe forms ofdisease than others (hypervirulent), and others rarely cause disease atall. Seven hypervirulent lineages are recognised, namely subgroups I,III and IV-1, ET-5 complex, ET-37 complex, A4 cluster and lineage 3.These have been defined by multilocus enzyme electrophoresis (MLEE), butmultilocus sequence typing (MLST) has also been used to classifymeningococci [ref. 41].

MODES FOR CARRYING OUT THE INVENTION

1. Meningococcal Saccharide Compositions for Human IntramuscularAdministration

Oligosaccharide conjugates from MenC, MenW135, MenY and, optionally,MenA were prepared as disclosed in reference 7. These were used toprepare individual 0.5 ml doses of the following six compositions(amounts per 0.5 ml dose):

Component A* B C* D* E* F Serogroup A oligosaccharide- μg 10 0 10 5 2.50 CRM₁₉₇ conjugate Serogroup C oligosaccharide- μg 10 10 5 5 2.5 10CRM₁₉₇ conjugate Serogroup W135 oligosaccharide- μg 10 10 5 5 2.5 0CRM₁₉₇ conjugate Serogroup Y oligosaccharide- μg 10 10 5 5 2.5 0 CRM₁₉₇conjugate Aluminium phosphate adjuvant mg 0.3 Sodium chloride mg 4.5Mannitol mg 7.5 Sodium phosphate monobasic (pH 7.6) mg 0.69 Potassiumdihydrogen phosphate mg 0.34 Tween ™ 80 mg 0.025 *The serogroup Acomponent was in lyophilised form and was diluted with a CWY liquidcomposition to give the final ACWY composition.

These vaccines are administered by intramuscular injection in the thighregion to toddlers aged 12-16 months, either in a single dose (which iseffective for Menjugate™ in children >12 months) or with a secondinjection 4 weeks later. Serum BCA and IgG can be comparedpre-vaccination and post-vaccination (e.g at 4 weeks, and then at 8weeks if two doses are received).

2. Two-Vial Composition

Conjugates for human use were prepared in two separate vials. Vial 1contained a lyophilised powder of MenA conjugate, with sucrose andpotassium dihydrogen phosphate. Vial 2 contained the MenC, MenW135 andMenY conjugates, with sodium chloride, polysorbate 80, sodium phosphatebuffer, and an optional aluminium phosphate adjuvant, which is presentin suspension. Prior to use, vial 1 is reconstituted with 0.6 ml liquidfrom vial 2, to give 0.5 ml available for administration.

Three doses were prepared. In reconstituted form, vaccines containedantigens as follows:

Component Quantity per 0.5 ml dose Serogroup A conjugate 10 μgsaccharide + 12.5-33 μg CRM₁₉₇ OR 5 μg saccharide + 6.25-16.5 μg CRM₁₉₇OR 2.5 μg saccharide + 3.125-8.25 μg CRM₁₉₇ Serogroup C conjugate 10 μgsaccharide + 12.5-25 μg CRM₁₉₇ OR 5 μg saccharide + 6.25-12.5 μg CRM₁₉₇OR 2.5 μg saccharide + 3.125-6.25 μg CRM₁₉₇ Serogroup W135 10 μgsaccharide + 6.6-20 μg CRM₁₉₇ conjugate OR 5 μg saccharide + 3.3-10 μgCRM₁₉₇ OR 2.5 μg saccharide + 1.65-5 μg CRM₁₉₇ Serogroup Y conjugate 10μg saccharide + 6.6-20 μg CRM₁₉₇ OR 5 μg saccharide + 3.3-10 μg CRM₁₉₇OR 2.5 μg saccharide + 1.65-5 μg CRM₁₉₇

In reconstituted form, vaccines contained other components as follows:

Component Quantity per 0.5 ml dose Aluminium phosphate adjuvant 0.3 mgas Al³⁺ zero Sodium dihydrogen phosphate 1 mM 2.5 mM Disodium hydrogenphosphate dihydrate 9 mM 7.5 mM Sodium phosphate buffer 10 mM Potassiumdihydrogen phosphate 5 mM Tween ™ 80 (surfactant) 0.025 mg Sodiumchloride (tonicity) 4.5 mg Sucrose (lyophilisation & tonicity) 12.5 mgWater for injection To final volume

Six vaccines were thus available—three different doses (10, 20 or 40 μgtotal saccharide), each with or without aluminium phosphate adjuvant.

The adjuvanted vaccine with the highest saccharide dose was administeredto healthy human subjects aged 18-45. For comparison, control subjectsreceived either (a) the vial 1 (reconstituted in buffer) and vial 2products in different arms at the same time, or (b) Mencevax™. Eachpatient group contains 30 people.

Blood was collected before and 28 days after vaccination to evaluate theimmune response and to collect laboratory safety parameters (completeblood count, blood chemistry analyses, liver and renal function testsand urinalysis). The vaccine was well tolerated, with no unexpectedadverse reactions. No significant abnormal changes in laboratoryparameters occurred during the study.

Serogroup A, C, W-135, Y specific SBA and IgG (measured by ELISA) weredetermined in the serum samples. SBA titres were expressed as thereciprocal of the final serum dilution giving ≥50% killing at 60minutes. For IgG measurement, a modified ELISA was performed to assayhigh avidity antibodies. For the detection of functional antibodies,SBAs with two different exogenous sources of complement were used: ababy rabbit complement source and a human complement source.

The high avidity IgG results were as follows (mean GMC (μg/mL) with 95%confidence intervals):

Vaccine Group Serogroup Serum ACWY A + CWY Mencevax ™ A Pre 0.67(0.3-1.2) 0.85 (0.4-1.5) 0.45 (0.2-0.8) Post   10 (6.6-16)   14 (8.8-22) 9.8 (6.2-15) C Pre 0.21 (0.1-0.3) 0.13 (0.07-0.2) 0.16 (0.1-0.2) Post 7.7 (4.7-13)  5.2 (3.19-8.46)  8.5 (5.2-14) W-135 Pre 0.21 (0.1-0.3) 0.2 (0.1-0.3) 0.29 (0.19-0.4) Post   12 (6.5-21)   9 (5.5-18)  6.7(3.7-12) Y Pre 0.35 (0.2-0.5) 0.31 (0.1-0.5) 0.57 (0.3-0.9) Post   18(12-29)   21 (13-33)   20 (12-31)

The SBA results were as follows (% responders and mean GMT, both with95% CI):

Rabbit Complement SBA Human Complement SBA Sero- Titres Titres groupVaccine Group ≥1:128 (%) GMT ≥1:4 (%) GMT A ACWY 93 (78-99) 989(558-1754) 90 (73-98) 42 (23-76)  A + CWY 97 (83-100) 2566 (1448-4549) 97 (83-100) 66 (36-119) MENCEVAX ™ 100 (88-100) 3132 (1767-5552) 83(65-94) 28 (15-50)  C ACWY  97 (82-100) 4480 (2455-8176) 100 (88-100)213 (106-427) A + CWY 100 (88-100) 3794 (2100-6855) 100 (88-100) 162(80-325) MENCEVAX ™ 93 (78-99) 3829 (2119-6918) 100 (88-100) 223(111-448) W-135 ACWY 100 (88-100) 10343 (5988-17865) 100 (88-100) 248(123-500) A + CWY 100 (88-100) 10376 (6007-17923) 93 (78-99) 142(71-287)  MENCEVAX ™ 100 (88-100)  6795 (3934-11737)  97 (83-100) 99(49-199) Y ACWY 100 (88-100)  22075 (14689-33175) 100 (88-100) 263(151-457) A + CWY 100 (88-100)  24034 (15993-36120) 100 (88-100) 162(194-588) MENCEVAX ™ 100 (88-100) 14630 (9735-21987) 100 (88-100) 198(114-344)

For each serogroup and in each vaccine group (ACWY, A+CWY and MENCEVAX™control) the high avidity ELISA anti-capsular IgG GMCs and the SBA GMTmeasured with both rabbit and human complement assay, increased afterinjection. At day 29 after vaccine injection, the percentage of subjectswith human complement SBA titers ≥1:4 for each serogroup ranged between90%-100% in the conjugate vaccines and between 83%-100% in the controlgroup. Using the rabbit complement source, the percentage of subjectswith SBA titers ≥1:128 for each serogroup ranged between 93%-100% forthe conjugate vaccines and between 90%-100% for the control group.

Overall, immune responses (GMC and GMT) were better in the conjugategroups than in the MENCEVAX™ control group. The improvement wasparticularly seen for serogroup W-135. The conjugate vaccines of theinvention are thus safe, well tolerated and induce functional immuneresponses equal to or better than those observed following immunisationwith a licensed tetravalent polysaccharide vaccine.

3. Use of Modified MenA Saccharide

Capsular polysaccharide was purified from MenA and was hydrolysed togive MenA oligosaccharide. The polysaccharide (2 g) was hydrolyzed at50° C. in 50 mM sodium acetate buffer, pH 4.75, at a polysaccharideconcentration of 10 mg/mL for about 4 hours [73]. After hydrolysis, thesolution was dried by rotary evaporation.

The oligosaccharide was activated using the following reaction scheme:

The oligosaccharide was dissolved in DMSO to give a saccharideconcentration of 10 mg/mL. According to a molar ratio ofoligosaccharide:CDI being 1:20, 21.262 g of CDI was then added and thereaction mixture stirred for 16 hours at room temperature. The resultingMenA-CDI compound was purified by selective precipitation in a 80:20(v/v) acetone:DMSO mixture followed by centrifugation. The efficiency ofthe activation reaction was calculated to be about 67.9% by determiningthe ratio of free imidazole to bonded imidazole.

In the second reaction step, the MenA-CDI oligosaccharide wassolubilised in DMSO at a saccharide concentration of about 10 mg/mL.According to a molar ratio of MenA-CDI unit:DMA being 1:100, 36.288 g of99% dimethylamine hydrochloride (i.e. R¹ & R²=Me) was added and thereaction mixture stirred for 16 hours at room temperature. The reactionproduct was freeze-dried and re-solubilised in 10 mg/mL water solution.

To remove the low molecular weight reaction reagent (in particular thedimethylamine (DMA)) from the oligosaccharide preparation, a dialysisstep was performed through a 3.5 kDa MWCO membrane (Spectra/Por™). Fourdialysis steps were carried out: (i) 16 hours against 2 L of 1 M sodiumchloride (dialysis factor 1:20), (ii) 16 hours against 2 L of 0.5 Msodium chloride (dialysis factor 1:20), (iii) and (iv) 16 hours against2 L of WFI (dialysis factor 1:20). To improve the purification adiafiltration step was also performed through a 1 kDa MWCO membrane(Centricon™)

The purified MenA-CDI-DMA product was buffered at pH 6.5 in 25 mML-histidine (Fluka™)

For preparing conjugates of the modified MenA saccharide (MenA-CDI-DMA),the overall process was as follows:

-   -   hydrolysis of the polysaccharide to give oligosaccharide        fragments    -   sizing of the oligosaccharide fragments    -   reductive amination of terminal aldehyde groups on the sized        oligosaccharides    -   protection of terminal —NH₂ groups by Fmoc group before the CDI        reaction    -   intrinsic de-protection of —NH₂ groups during the DMA reaction    -   activation of terminal —NH₂ groups by SIDEA        (N-hydroxysuccinimide adipic acid)    -   covalent attachment to CRM₁₉₇ protein.

The modified MenA oligosaccharide conjugate was much more resistant tohydrolysis than its natural counterpart at elevated temperatures. After28 days at 37° C., for instance, the percentage of released saccharideis 6.4% for the modified oligosaccharide vs. 23.5% for the naturalantigen. Moreover, the titres induced by the modified oligosaccharidesare not significantly lower than those obtained using the native sugarstructures.

The modified MenA conjugate is combined with MenC, MenW135 and MenYconjugates as a substitute for the conjugate of unmodifiedoligosaccharide.

4. Addition of MenB Antigens

Prior to reconstitution of the lyophilised MenA conjugate as describedabove, MenB antigens ΔG287-953 (SEQ ID NO: 7), 936-ΔG741 (SEQ ID NO: 8)and NadA (SEQ ID NO: 2) are added to the highest-dose liquid C-W135-Ymixture to give a final concentration of 20 μg/dose of each of the threepolypeptides. The reconstituted vaccine thus contains the followingantigens:

Component Quantity per 0.5 ml dose Serogroup A conjugate 10 μgsaccharide + 12.5-33 μg CRM₁₉₇ Serogroup C conjugate 10 μg saccharide +12.5-25 μg CRM₁₉₇ Serogroup W135 conjugate 10 μg saccharide + 6.6-20 μgCRM₁₉₇ Serogroup Y conjugate 10 μg saccharide + 6.6-20 μg CRM₁₉₇ΔG287-953 20 μg polypeptide 936-ΔG741 20 μg polypeptide NadA 20 μgpolypeptide

5. Addition of Hib Antigen

Lyophilised HbOC conjugate is mixed with the lyophilised MenA conjugateand both are reconstituted together by liquid C-W135-Y mixture to givefollowing vaccine:

Component Quantity per 0.5 ml dose Serogroup A conjugate 10 μgsaccharide + 12.5-33 μg CRM₁₉₇ Serogroup C conjugate 10 μg saccharide +12.5-25 μg CRM₁₉₇ Serogroup W135 conjugate 10 μg saccharide + 6.6-20 μgCRM₁₉₇ Serogroup Y conjugate 10 μg saccharide + 6.6-20 μg CRM₁₉₇ HbOCHib conjugate 10 μg saccharide + 2-5 μg CRM₁₉₇

6. Addition of Pneumococcal Antigens

Prior to reconstitution of the lyophilised MenA conjugate as describedabove, pneumococcal conjugate antigens are added to the medium-doseliquid C-W135-Y mixture to give a final concentration of 2 μg/dose ofeach of the serotypes (double for serotype 6B). The reconstitutedvaccine thus contains the following antigens:

Component Quantity per 0.5 ml dose Serogroup A conjugate 5 μgsaccharide + 6.25-16.5 μg CRM₁₉₇ Serogroup C conjugate 5 μg saccharide +6.25-12.5 μg CRM₁₉₇ Serogroup W135 conjugate 5 μg saccharide + 3.3-10 μgCRM₁₉₇ Serogroup Y conjugate 5 μg saccharide + 3.3-10 μg CRM₁₉₇Pneumococcus serotype 4 2 μg saccharide + 2.5 μg CRM₁₉₇ conjugatePneumococcus serotype 9V 2 μg saccharide + 2.5 μg CRM₁₉₇ conjugatePneumococcus serotype 14 2 μg saccharide + 2.5 μg CRM₁₉₇ conjugatePneumococcus serotype 18C 2 μg saccharide + 2.5 μg CRM₁₉₇ conjugatePneumococcus serotype 19F 2 μg saccharide + 2.5 μg CRM₁₉₇ conjugatePneumococcus serotype 23F 2 μg saccharide + 2.5 μg CRM₁₉₇ conjugatePneumococcus serotype 6B 4 μg saccharide + 5 μg CRM₁₉₇ conjugate

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES The Contents of which are Hereby Incorporated in Full

-   [1] Chapter 28 of Vaccines (Plotkin & Orenstein) 3rd edition (1999)    ISBN 0-7216-7443-7.-   [2] Armand et al. (1982) J. Biol. Stand. 10:335-339.-   [3] Cadoz et al. (1985) Vaccine 3:340-342.-   [4] MMWR (1997) 46(RR-5) 1-10.-   [5] Baldaic et al. (1983) Infect. Immun. 42:599-604.-   [6] WO02/058737.-   [7] WO03/007985.-   [8] Rennels et al. (2002) Pediatr Infect Dis J 21:978-979.-   [9] Campbell et al. (2002) J Infect Dis 186:1848-1851.-   [10] Costantino et al. (1992) Vaccine 10:691-698.-   [11] Jones (2001) Curr Opin Investig Drugs 2:47-49.-   [12] WO02/00249.-   [13] WO97/28273.-   [14] Lieberman et al. (1996) JAMA 275:1499-1503.-   [15] Frash (1990) p. 123-145 of Advances in Biotechnological    Processes vol. 13 (eds. Mizrahi & Van Wezel)-   [16] Inzana (1987) Infect. Immun. 55:1573-1579.-   [17] Kandil et al. (1997) Glycoconj J 14:13-17.-   [18] Berkin et al. (2002) Chemistry 8:4424-4433.-   [19] WO03/080678.-   [20] WO98/08543.-   [21] Tettelin et al. (2000) Science 287:1809-1815.-   [22] Pizza et al. (2000) Science 287:1816-1820.-   [23] WO99/24578.-   [24] WO99/36544.-   [25] WO99/57280.-   [26] WO00/22430.-   [27] WO00/66791.-   [28] WO00/66741.-   [29] WO01/64920.-   [30] WO01/64922.-   [31] WO03/020756.-   [32] UK patent applications 0223741.0, 0305831.0 & 0309115.4, and    international application PCT/IB03/04848.-   [33] Comanducci et al. (2002) J. Exp. Med. 195:1445-1454.-   [34] WO03/010194.-   [35] Parkhill et al. (2000) Nature 404:502-506.-   [36] International patent application PCT/IB03/06320.-   [37] WO03/063766.-   [38] Masignani et al. (2003) J Exp Med 197:789-799.-   [39] http://neisseria.org/nm/typing/mlst/-   [40] Pettersson et al. (1994) Microb Pathog 17(6):395-408.-   [41] Maiden et al. (1998) PNAS USA 95:3140-3145.-   [42] Welsch et al. (2002) Thirteenth International Pathogenic    Neisseria Conference, Norwegian Institute of Public Health, Oslo,    Norway; Sep. 1-6, 2002. Genome-derived antigen (GNA) 2132 elicits    protective serum antibodies to groups B and C Neisseria meningitidis    strains.-   [43] Santos et al. (2002) Thirteenth International Pathogenic    Neisseria Conference, Norwegian Institute of Public Health, Oslo,    Norway; Sep. 1-6, 2002. Serum bactericidal responses in rhesus    macaques immunized with novel vaccines containing recombinant    proteins derived from the genome of N. meningitidis.-   [44] Lindberg (1999) Vaccine 17 Suppl 2:S28-36.-   [45] Buttery & Moxon (2000) J R Coll Physicians Lond 34:163-168.-   [46] Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-33,    vii.-   [47] Goldblatt (1998) J. Med. Microbiol. 47:563-567.-   [48] European patent 0477508.-   [49] U.S. Pat. No. 5,306,492.-   [50] WO98/42721.-   [51] Dick et al. in Conjugate Vaccines (eds. Cruse et al.) Karger,    Basel, 1989, 10:48-114.-   [52] Hermanson Bioconjugate Techniques, Academic Press, San    Diego (1996) ISBN: 0123423368.-   [53] Kanra et al. (1999) The Turkish Journal of Paediatrics    42:421-427.-   [54] Ravenscroft et al. (2000) Dev Biol (Basel) 103: 35-47.-   [55] WO97/00697.-   [56] WO96/37222; U.S. Pat. No. 6,333,036.-   [57] Watson (2000) Pediatr Infect Dis J 19:331-332.-   [58] Rubin (2000) Pediatr Clin North Am 47:269-285, v.-   [59] Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207.-   [60] Zielen et al. (2000) Infect. Immun. 68:1435-1440.-   [61] Darkes & Plosker (2002) Paediatr Drugs 4:609-630.-   [62] Tettelin et al. (2001) Science 293:498-506.-   [63] Hoskins et al (2001) J Bacteriol 183:5709-5717.-   [64] Rappuoli (2000) Curr Opin Microbiol 3:445-450-   [65] Rappuoli (2001) Vaccine 19:2688-2691.-   [66] Masignani et al. (2002) Expert Opin Biol Ther 2:895-905.-   [67] Mora et al. (2003) Drug Discov Today 8:459-464.-   [68] Wizemann et al. (2001) Infect Immun 69:1593-1598.-   [69] Rigden et al. (2003) Crit Rev Biochem Mol Biol 38:143-168.-   [70] WO02/22167.-   [71] Nilsson & Svensson (1979) Carbohydrate Research 69: 292-296)-   [72] Ravenscroft et al. (1999) Vaccine 17:2802-2816.-   [73] Costantino et al. (1999) Vaccine 17:1251-1263.-   [74] Ramsay et al. (2001) Lancet 357(9251):195-196.-   [75] Anonymous (January 2002) Research Disclosure, 453077.-   [76] Anderson (1983) Infect Immun 39(1):233-238.-   [77] Anderson et al. (1985) J Clin Invest 76(1):52-59.-   [78] EP-A-0372501.-   [79] EP-A-0378881.-   [80] EP-A-0427347.-   [81] WO93/17712-   [82] WO94/03208.-   [83] WO98/58668.-   [84] EP-A-0471177.-   [85] WO91/01146-   [86] Falugi et al. (2001) Eur J Immunol 31:3816-3824.-   [87] EP-A-0594610.-   [88] WO00/56360.-   [89] WO02/091998.-   [90] WO01/72337-   [91] WO00/61761.-   [92] WO99/42130-   [93] WO96/40242-   [94] Lees et al. (1996) Vaccine 14:190-198.-   [95] WO95/08348.-   [96] U.S. Pat. No. 4,882,317-   [97] U.S. Pat. No. 4,695,624-   [98] Porro et al. (1985) Mol Immunol 22:907-919.-   [99] EP-A-0208375-   [100] WO00/10599-   [101] Gever et al. Med. Microbiol. Immunol, 165: 171-288 (1979).-   [102] U.S. Pat. No. 4,057,685.-   [103] U.S. Pat. Nos. 4,673,574; 4,761,283; 4,808,700.-   [104] U.S. Pat. No. 4,459,286.-   [105] U.S. Pat. No. 4,965,338-   [106] U.S. Pat. No. 4,663,160.-   [107] U.S. Pat. No. 4,761,283-   [108] U.S. Pat. No. 4,356,170-   [109] Lei et al. (2000) Dev Biol (Basel) 103:259-264.-   [110] WO00/38711; U.S. Pat. No. 6,146,902.-   [111] WO03/009869.-   [112] Vaccine Design . . . (1995) eds. Powell & Newman. ISBN:    030644867X. Plenum.-   [113] WO00/23105.-   [114] U.S. Pat. No. 5,057,540.-   [115] WO96/33739.-   [116] EP-A-0109942.-   [117] WO96/11711.-   [118] WO00/07621.-   [119] Barr et al. (1998) Advanced Drug Delivery Reviews 32:247-271.-   [120] Sjolanderet et al. (1998) Advanced Drug Delivery Reviews    32:321-338.-   [121] Niikura et al. (2002) Virology 293:273-280.-   [122] Lenz et al. (2001) J Immunol 166:5346-5355.-   [123] Pinto et al. (2003) J Infect Dis 188:327-338.-   [124] Gerber et al. (2001) Virol 75:4752-4760.-   [125] WO03/024480-   [126] WO03/024481-   [127] Gluck et al. (2002) Vaccine 20:B10-B16.-   [128] EP-A-0689454.-   [129] Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-2278.-   [130] Evans et al. (2003) Expert Rev Vaccines 2:219-229.-   [131] Meraldi et al. (2003) Vaccine 21:2485-2491.-   [132] Pajak et al. (2003) Vaccine 21:836-842.-   [133] Kandimalla et al. (2003) Nucleic Acids Research 31:2393-2400.-   [134] WO02/26757.-   [135] WO99/62923.-   [136] Krieg (2003) Nature Medicine 9:831-835.-   [137] McCluskie et al. (2002) FEMS Immunology and Medical    Microbiology 32:179-185.-   [138] WO98/40100.-   [139] U.S. Pat. No. 6,207,646.-   [140] U.S. Pat. No. 6,239,116.-   [141] U.S. Pat. No. 6,429,199.-   [142] Kandimalla et al. (2003) Biochemical Society Transactions 31    (part 3):654-658.-   [143] Blackwell et al. (2003) J Immunol 170:4061-4068.-   [144] Krieg (2002) Trends Immunol 23:64-65.-   [145] WO01/95935.-   [146] Kandimalla et al. (2003) BBRC 306:948-953.-   [147] Bhagat et al. (2003) BBRC 300:853-861.-   [148] WO03/035836.-   [149] WO95/17211.-   [150] WO98/42375.-   [151] Beignon et al. (2002) Infect Immun 70:3012-3019.-   [152] Pizza et al. (2001) Vaccine 19:2534-2541.-   [153] Pizza et al. (2000) Int J Med Microbiol 290:455-461.-   [154] Scharton-Kersten et al. (2000) Infect Immun 68:5306-5313.-   [155] Ryan et al. (1999) Infect Immun 67:6270-6280.-   [156] Partidos et al. (1999) Immunol Lett 67:209-216.-   [157] Peppoloni et al. (2003) Expert Rev Vaccines 2:285-293.-   [158] Pine et al. (2002) J Control Release 85:263-270.-   [159] Domenighini et al. (1995) Mol Microbiol 15:1165-1167.-   [160] WO99/40936.-   [161] WO99/44636.-   [162] Singh et al] (2001) J Cont Release 70:267-276.-   [163] WO99/27960.-   [164] U.S. Pat. No. 6,090,406-   [165] U.S. Pat. No. 5,916,588-   [166] EP-A-0626169.-   [167] WO99/52549.-   [168] WO01/21207.-   [169] WO01/21152.-   [170] Andrianov et al. (1998) Biomaterials 19:109-115.-   [171] Payne et al. (1998) Adv Drug Delivery Review 31:185-196.-   [172] Stanley (2002) Clin Exp Dermatol 27:571-577.-   [173] Jones (2003) Curr Opin Investig Drugs 4:214-218.-   [174] WO96/14086.-   [175] Vaccines (eds. Plotkin & Mortimer), 1988. ISBN: 0-7216-1946-0-   [176] Gustafsson et al. (1996) N. Engl. J. Med. 334:349-355.-   [177] Rappuoli et al. (1991) TIBTECH 9:232-238.-   [178] Bell (2000) Pediatr Infect Dis J 19:1187-1188.-   [179] Iwarson (1995) APMIS 103:321-326.-   [180] Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.-   [181] WO93/24148.-   [182] WO02/09643.-   [183] Katial et al. (2002) Infect Immun 70:702-707.-   [184] WO01/52885.-   [185] European patent 0301992.-   [186] Bjune et al. (1991) Lancet 338(8775):1093-1096.-   [187] Fukasawa et al. (1999) Vaccine 17:2951-2958.-   [188] WO02/09746.-   [189] Rosenqvist et al. (1998) Dev. Biol. Stand. 92:323-333.-   [190] WO01/09350.-   [191] European patent 0449958.-   [192] EP-A-0996712.-   [193] EP-A-0680512.-   [194] WO02/062378.-   [195] WO99/59625.-   [196] U.S. Pat. No. 6,180,111.-   [197] WO01/34642.-   [198] WO03/051379.-   [199] U.S. Pat. No. 6,558,677-   [200] PCT/IB03/04293.-   [201] WO02/062380.-   [202] WO00/25811.-   [203] Peeters et al. (1996) Vaccine 14:1008-1015.-   [204] Vermont et al. (2003) Infect Immun 71:1650-1655.-   [205] Sutter et al. (2000) Pediatr Clin North Am 47:287-308.-   [206] Zimmerman & Spann (1999) Am Fam Physician 59:113-118, 125-126.-   [207] Charalambous & Feavers (2001) J Med Microbiol 50:937-939.-   [208] Westerink (2001) Int Rev Immunol 20:251-261.-   [209] Grothaus et al. (2000) Vaccine 18:1253-1263.-   [210] Paoletti et al. (2001) Vaccine 19:2118-2126.-   [211] WO00/56365.-   [212] Gennaro (2000) Remington: The Science and Practice of    Pharmacy. 20th ed ISBN: 0683306472-   [213] WO01/30390.

1-25. (canceled) 26: An injectable immunogenic composition comprisingcapsular saccharides from at least three serogroups A, C, W135 and Y ofNeisseria meningitidis, wherein the capsular saccharides areoligosaccharides and are conjugated to one or more carrier proteins, andwherein the composition comprises less than 50 ug meningococcalsaccharide per dose. 27: An injectable immunogenic compositioncomprising capsular saccharides from at least three serogroups A, C,W135 and Y of Neisseria meningitidis, wherein: the capsular saccharidesare oligosaccharides and are conjugated to one or more carrier proteins,and wherein the composition further comprises an antigen from one ormore of (a) serogroup B Neisseria meningitidis; (b) Haemophilusinfluenza type B; and (c) Streptococcus pneumoniae. 28: The compositionof claim 26 or claim 27, comprising antigens that protect againstserogroups C, W135 and Y of Neisseria meningitidis. 29: The compositionof claim 28, further comprising an antigen that protects againstserogroup A of Neisseria meningitidis. 30: The composition of claim 29,wherein the antigen that protects against serogroup A of Neisseriameningitidis is a modified serogroup A capsular saccharide in which oneor more hydroxyl groups have been replaced with blocking groups. 31: Thecomposition of claim 28, further comprising an antigen that protectsagainst Haemophilus influenza type B. 32: The composition of claim 28,further comprising one or more antigens that protect against serogroup Bof Neisseria meningitidis. 33: The composition of claim 32, where theone or more antigens can induce, after administration to a subject, abacterial antibody response in that subject against two or more ofhypervirulent lineages A4, ET5 and lineage 3 of Neisseria meningitidisserogroup B. 34: The composition of claim 32, wherein the one or moreantigens comprise one or more of (a) a NadA protein in oligomeric form;(b) a 741 protein; (c) a 936 protein; (d) a 953 protein; and (e) a 287protein. 35: The composition of claim 34, wherein the one or moreantigens comprise (a) a NadA protein in oligomeric form; (b) a 741protein; (c) a 936 protein; (d) a 953 protein; and (e) a 287 protein.36: The composition of claim 34, comprising a first polypeptidecomprising amino acid sequence SEQ ID NO:2, a second polypeptidecomprising amino acid sequence SEQ ID NO: 7 and a third polypeptidecomprising amino acid sequence SEQ ID NO:
 8. 37: The composition ofclaim 28 further comprising one or more antigens that protect againstStreptococcus pneumoniae. 38: The composition of claim 37, wherein theone or more antigens comprise a capsular saccharide from Streptococcuspneumoniae. 39: The composition of claim 38, comprising capsularsaccharides from between 5 and 11 different serotypes of Streptococcuspneumoniae. 40: The composition of claim 37, wherein the one or moreantigens comprise polypeptides from Streptococcus pneumoniae. 41: Thecomposition of claim 40, wherein the composition comprises one or moreof the Streptococcus pneumoniae antigens PhtA, PhtD, PhtB, PhtE, SpsA,LytB, LytC, LytA, Sp125, Sp101, Sp128 and Sp130. 42: The composition ofclaim 28, wherein saccharide antigens in the composition are conjugatedto carrier proteins. 43: The composition of claim 42, wherein saccharideantigens in the composition are oligosaccharides conjugated to CRM197protein, protein D of Haemophilus influenza, tetanus toxoid ordiphtheria toxoid. 44: The composition of claim 42, wherein theconjugates have a saccharide:protein ratio (w/w) between 1:5 and 5:1.45: The composition of claim 28 comprising less than 25 ug meningococcalsaccharide per dose. 46: The composition of claim 28, further comprisingan aluminum salt adjuvant. 47: A kit comprising lyophilized capsularsaccharides from Neisseria meningitidis serogroup A and liquid capsularsaccharides from Neisseria meningitidis serogroup C, W135 and Y 48: Thekit of claim 47, wherein the capsular saccharides from Neisseriameningitidis serogroup A are modified serogroup A capsular saccharidesin which one or more hydroxyl groups have been replaced with blockinggroups. 49: A method of preparing the composition of claim 28 comprisingmixing lyophilized capsular saccharides from Neisseria meningitidisserogroup A with liquid capsular saccharides from Neisseria meningitidisserogroup C, W135 and Y. 50: The method of claim 49, wherein thecapsular saccharides from Neisseria meningitidis serogroup A aremodified serogroup A capsular saccharides in which one or more hydroxylgroups have been replaced with blocking groups. 51: A method ofgenerating an immune response in a human comprising injecting the humanwith the composition of claim
 28. 52: A method of generating an immuneresponse in a human comprising injecting the human with capsularconjugated oligosaccharides from serogroups A, C, W135 and Y ofNeisseria meningitidis and one or more antigens chosen from serogroup BNeisseria meningitidis; Haemophilus influenza type B; and Streptococcuspneumoniae.